JP2023078366A - Radiolabeling of polypeptide - Google Patents

Abstract

To provide a method for labelling a polypeptide by radioactive metal ions by using click chemistry.SOLUTION: A method for labelling polypeptide by radioactive metal ions includes: providing a modified polypeptide including a polypeptide covalently bonded with a first click reaction partner; and providing a radioactive complex including the radioactive metal ions associated with a chelated portion, herein, the chelated portion includes chelator covalently bonded with a second click reaction partner; and bringing the modified polypeptide in contact with the radioactive complex under the condition enabling the polypeptide to be labelled by the radioactive metal ions by allowing the first click reaction partner to react with the second click reaction partner.SELECTED DRAWING: None

Description

(REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY)
This application is filed under "Sequence Listing
which is submitted electronically via EFS-Web as a Sequence Listing in ASCII format with the file name . The Sequence Listing submitted via EFS-Web is part of this specification and is hereby incorporated by reference in its entirety.

(Cross reference to related applications)
This application is entitled pursuant to 35 U.S.C. incorporated into.

(Field of Invention)
The present invention relates to methods of radiolabeling polypeptides such as antibodies. Specifically, the invention relates to methods of labeling polypeptides with radioactive metal ions using click chemistry. The invention also relates to pharmaceutical compositions and uses of radiolabeled polypeptides.

Alpha-emitting radionuclides hold great promise for cancer therapy because of their combination of high energy and short-range action, most of which are localized to tumor cells and offer potent killing potential (Kim, Y. S. and M. W. Brechbiel, An overvie
w of targeted alpha therapy. Tomorrow Biol,
2012.33(3): p. 573-90). Targeted delivery of alpha-emitters using antibodies, scaffold proteins, small molecule ligands, aptamers, or other binding moieties specific for cancer antigens provides a method for selective delivery of radionuclides to tumors. methods to enhance their potency and mitigate off-target effects. In common practice, the binding moiety is attached to a chelator that binds to the alpha-emitting metal to form a radioactive complex. Many such examples use monoclonal antibodies (mAbs) as targeting ligands to produce what are known as radioimmunoconjugates.

Actinium-225 ( ²²⁵ Ac) is an alpha-radioactive isotope of particular interest for medical applications (Miederer et al., Realizing the pot
final of the Actinium-225 radionuclei
generator in targeted alpha particles the
rapy applications. Adv Drug Deliv Rev, 200
8.60(12):71-82). The 10-day half-life of ²²⁵ Ac is long enough to facilitate radioconjugate formation, but short enough to match the circulating pharmacokinetics of delivery vehicles such as antibodies. Radioimmunoconjugates of ²²⁵ Ac are therefore of particular interest. In addition, ²²⁵ Ac increases potency by decaying in a series of steps that eventually emit four alpha particles before reaching the stable isotope ²⁰⁹ Bi. Another radioisotope for medical use is lutetium-177 ( ¹⁷⁷ Lu), which emits both gamma radiation suitable for imaging and medium energy beta radiation suitable for radiotherapy. ^177Lu -labeled peptides demonstrate a reduction in normal tissue damage, and ^177Lu -labeling has been shown to allow the use of a single radiolabeled agent for both therapy and imaging ( Kwekkeboom DJ
, et al. [177Lu-DOTAOTyr3]octreotate:compa
rison with [111In-DTPAo] octreotide in pat
ents. Eur J Nucl Med. 2001; 28:p. 1319-1325
). Other radioisotopes used in therapeutic applications include, for example, beta-emitters or alpha-emitters such as thorium, radium, ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁷⁷ As,
⁸⁹ Sr, ⁹⁰ Y, ⁹⁹ Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹³¹ I, ¹⁵³
Sm, ¹⁵⁹ Gd, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁸⁶ Re, ¹⁸⁸ Re, ^1
94 Ir, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹² Pb, ²¹² Bi, ²¹³ Bi
, ²²³ Ra, ²⁵⁵ Fm, and ²²⁷ Th. Other radioisotopes used for imaging applications include, for example, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸
Gamma-emitting radioisotopes such as ⁹ Zr and ¹¹¹ In are included.

Previous clinical and preclinical programs for actinium chelation,1,
4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) has been used. However, DOTA chelation of actinium is known to be potentially difficult (Deal, KA, et al., Improved in
vivo stability of actinium-225 macrocyc
lic complexes. J Med Chem, 1999.42(15):p. 2
988-92), often requiring harsh conditions or high levels of DOTA per antibody. As a result, two
Two different approaches have been utilized, each with its own drawbacks.

A “two-step” method was first developed involving two chemical steps involving actinium (M
cDevitt, M.; R. , et al. , Tumor therapy with t
targeted atomic nanogenerators. Science, 20
01.294 (5546): p. 1537-40). ²²⁵ Ac at pH 4.5-5 using 2M acetate buffer for 30 minutes at 55-60°C with high radiochemical yields (approximately 9
5%) with bifunctional chelator (BFC) DOTA-isothiocyanate (DOTA-SCN
) to chelate. ²²⁵ Ac DOTA-SCN was then reacted with the targeting antibody to generate a radioimmunoconjugate. A major drawback of the two-step method is that approximately 90% of the SCN cannot tolerate the labeling conditions, resulting in approximately 90% of the input ²²⁵ Ac being conjugated to an unreacted form of DOTA that cannot be conjugated to the antibody. It is that you are. This results not only in low yields (typically only about 10%) and high costs, but also in low specific radioactivity, which can limit the efficacy of the final conjugate.

A "one-step" method was recently developed for actinium (Maguire, WF.
, et al. , Efficient 1-step radio labeling o
f monoclonal antibodies to high specific
activity with 225Ac for alpha-particle
radioimmunotherapy of cancer. J Nucl Med,
2014.55 (9): p. 1492-8). This method has only one chemical reaction step involving actinium. DOTA-SCN was first conjugated to antibody. then ²²⁵ A
c was chelated to DOTA-mAb under mild conditions (37° C., pH 7.5) with radiochemical yields up to 80%. However, high levels of DOTA (approximately 10
above) to achieve high yields. High chelator:antibody ratio (C
AR), in this case species with a high DOTA:Ab ratio (DAR) are more likely to be immunoreactive, and moreover, the average DAR may be 10, whereas ²²⁵ Ac is higher than the average. may also be chelating to higher proportions of the population. This method therefore risks linking ²²⁵ Ac to at least the active fraction of the antibody-chelator complex.
Furthermore, the need to handle antibody and DOTA-mAb conjugates under metal-free conditions to avoid chelation of common metals such as iron, zinc, and copper poses significant challenges to the production process. .

Click chemistry, a chemical approach introduced by Sharpless in 2001, is a chemical process tailored to rapidly and reliably produce substances by linking together small amounts of units. For example, Kolb, Finn and Sha
rpress Angewandte Chemie International E
dition (2001) 40:2004-2021; Evans, Australia.
n Journal of Chemistry (2007) 60:384-395. Coupling reactions (some can be classified as "click chemistry")
Examples include, but are not limited to, formation of esters, thioesters, amides from activated acids or acyl halides (e.g., peptide coupling); nucleophilic substitution reactions (e.g., nucleophilic substitution of halides or strained ring systems ( azide-alkynes and Huisgen cycloaddition reactions (e.g., 1,3-dipolar cycloaddition reactions between azides and alkynes to form 1,2,3-triazole linkers); ); thioline addition reactions; imine formation; Diels-Alder reactions between tetrazines and trans-cyclooctene (TCO); and Michael addition reactions (eg, maleimide addition reactions).

Click chemistry reactions between alkynes and azides typically require the addition of a copper catalyst to promote the 1,3-cycloaddition reaction and are known as copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. It is However, the click chemistry reaction between cyclooctyne or a cyclooctyne derivative and an azide typically does not require the addition of a copper catalyst and instead uses a strain-promoted azide-alkyne cycloaddition reaction (SPAAC). p
(Debets, M. F., e.
tal. , Bioconjugation with strained alkenes
es and alkynes. Acc Chem Res, 2011.44(9):p
. 805-15).

Antibody-drug conjugates (ADC:
antibody-drug conjugate) has become a major focus area (Agarwal
, P. and C.I. R. Bertozzi, Site-specific antibo
dy-drug conjugates: the nexus of bioortho
global chemistry, protein engineering, and
drug development. Bioconjug Chem, 2015.26 (
2): p. 176-92). It is believed that similar safety and efficacy benefits can be achieved with radioimmunoconjugates.

As noted above, there remains a need in the art for efficient methods of producing stable radioimmunoconjugates with high specific activity and high yield.

The present invention fills this need by providing a method of using click chemistry to radiolabel polypeptides such as antibodies. The methods of the present invention require reduced use of radiometals, requiring only metal-free conditions in the steps used to generate the initial radioactive complexes, while azide-modified antibodies and chelating moieties comprising alkyne groups. Radioactive complexes containing radiometal ions associated with are used in click chemistry reactions to generate stable radioimmunoconjugates with low chelator:antibody ratios (CAR) and high radiochemical yields. The method of the present invention simplifies previous methods for producing radioimmunoconjugates while increasing safety, efficacy and uniformity.

In one general aspect, the invention relates to a method of labeling a polypeptide with a radioactive metal ion, the method comprising:
a. providing a modified polypeptide comprising a polypeptide covalently attached to a first click reaction partner;
b. providing a radioactive complex comprising a radioactive metal ion associated with a chelating moiety;
wherein the chelating moiety comprises a chelating agent covalently attached to the second click reaction partner;
c. contacting the modified polypeptide with a radioactive complex under conditions that allow the first click reaction partner to react with the second click reaction partner to label the polypeptide with a radiometal ion; include.

In another general aspect, the invention relates to pharmaceutical compositions comprising a radiolabeled polypeptide prepared by the method of the invention and a pharmaceutically acceptable carrier.

In another general aspect, the invention provides a method of treating a neoplastic disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention. to a method comprising:

In another general aspect, the invention comprises:
a. a modified polypeptide comprising a polypeptide covalently attached to a first click reaction partner;
b. a radioactive complex comprising a radioactive metal ion associated with a chelating moiety, the chelating moiety comprising a chelating agent covalently bound to a second click reaction partner,
Here, the combination or kit is used to label the polypeptide with radioactive metal ions.

In another general aspect, the invention relates to a therapeutic or diagnostic agent (“theranostic agent”) comprising a radiolabeled polypeptide prepared by the method of the invention.

The above "means of solving the problem" and the following "detailed description" will be better understood when read in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

The drawings are as follows.
1 shows a schematic diagram of radiolabeling of antibodies according to the method of the invention. FIG. A similar radiolabeling scheme is used when random conjugation is shown in the figure and the azide is site-specifically conjugated to a monoclonal antibody (mAb). Figure 2 shows a synthetic scheme for an improved two-step preparation of ^89Zr -DOTA-mAb via click chemistry, according to one embodiment of the present application. Cell binding of the In-111 radioimmunoconjugate is shown, with bound radioactivity increasing with increasing cell number. In particular, A shows the human prostate cancer cell line C4-2B (PSMA+, Transferrin B shows binding to human epidermoid carcinoma cell line A431 (EGFR+) by EGFR-binding antibodies, cetuximab and panitumumab In-111 radioimmunoconjugates according to embodiments of the present application, and these of the conjugates of the control (EGFR-) to the human AML cell line MOLM-13. Figure 2 shows the kinetics of cellular internalization of In-111 in human prostate cancer cell line C4-2B treated with an anti-PSMA mAb In-111 radioimmunoconjugate, according to one embodiment of the present application, surface bound In-111 It rapidly disappeared from the cell surface and was redistributed within the cell. We show the results of a mouse tumor xenograft study in which mice were implanted with human prostate cancer LNCaP cells and when tumors reached 100 mm ³ , radioactivity range or isotype control (for viral targets not present in this system radioconjugate) was administered. Mice were treated with a single dose of a click radiolabeled anti-PSMA mAb (“PSMB127”) actinium radioconjugate according to one embodiment of the present application of binding human IgG4 antibody). Specifically, the tumor volume for each group was shown and the size plotted until less than half of the group survived. We show the results of a mouse tumor xenograft study in which mice were implanted with human prostate cancer LNCaP cells and when tumors reached 100 mm ³ , radioactivity range or isotype control (for viral targets not present in this system radioconjugate) was administered. Mice were treated with a single dose of a click radiolabeled anti-PSMA mAb (“PSMB127”) actinium radioconjugate according to one embodiment of the present application of binding human IgG4 antibody). Specifically, survival curves for the control mAb group are shown. We show the results of a mouse tumor xenograft study in which mice were implanted with human prostate cancer LNCaP cells and when tumors reached 100 mm ³ , radioactivity range or isotype control (for viral targets not present in this system radioconjugate) was administered. Mice were treated with a single dose of a click radiolabeled anti-PSMA mAb (“PSMB127”) actinium radioconjugate according to one embodiment of the present application of binding human IgG4 antibody). Specifically, survival curves for the anti-PSMA mAb group are shown.

Various publications, articles and patents are cited or described in the background of the invention and throughout this specification. Each of these references is incorporated herein by reference in its entirety. Discussion of documents, operations, materials, devices, articles, etc. contained herein is intended to provide a context for the invention. Such discussion is not an admission that any or all of these items constitute prior art to any invention disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms cited herein shall have the meanings set forth herein. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth herein in their entirety.

As used in this specification and the appended claims, the singular forms "a", "an" and "the"
” includes plural referents unless the context clearly dictates otherwise.

Throughout this specification and the claims below, unless the context requires otherwise, the term "comprising"
"comprises" and variations such as "comprises" and "comprising"
It will be understood to mean including the specified integers or steps or groups of integers or steps, but not excluding any other integers or steps or groups of integers or steps. As used herein, the term "comprising" may be replaced by the term "containing" or "including," or when used herein,
It can also be replaced by the term "having".

As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel features of the claim. When used herein in connection with aspects or embodiments of the invention, the terms "comprising", "containing", "including" and "comprising" are used to vary the scope of the present disclosure. Any of the above terms of "comprising" can be replaced with the terms "consisting of" or "consisting essentially of".

As used herein, the conjunctive term "and/or" between multiple listed elements is
It is understood to include both individual and combined options. For example, if two elements are connected by "and/or", the first option refers to the applicability of the first element without the second element. A second option refers to the second element being applicable without the first element. A third option refers to the first and second elements being applicable together. It is understood that any one of these options will meet the requirements of the term "and/or" as used herein. Concurrent applicability of two or more of the options is also understood to be included in the meaning, thus satisfying the requirement of the term "and/or".

To assist the reader of this application, the description of the specification is divided into various paragraphs or sections, or directed to various embodiments of the application. These separations shall not be construed as separating one paragraph or section or the substance of an embodiment from another paragraph or section or the substance of an embodiment. On the contrary, those skilled in the art will appreciate that the description herein has broad application and encompasses all possible combinations of the various paragraphs, paragraphs, and sentences. Discussion of any embodiment is meant to be exemplary only and is not intended to suggest that the scope of the present disclosure, including the claims, is limited to these examples. .

Click Radiolabeling of Polypeptides In contrast to known procedures, the method of the present invention provides, for example, a subject in need thereof, such as, for example,
Provided are improved methods for the production of radioimmunoconjugates suitable for medical use in humans. In particular, the methods described herein include, but are not limited to, ²²⁵ Ac, ¹¹¹ In and ⁸
By providing a process for both high yield chelation and low DAR of metal ions, including ⁹ Zr, the major limitations of current methods are addressed. The present invention is also useful for radiolabeled diagnostic purposes (e.g. when labeled with ⁸⁹ Zr or ¹¹¹ In) or therapeutic purposes (e.g.
²²⁵ Ac), allowing the production of a single batch of azide-labeled polypeptides such as azide-mAb conjugates that can be used for production of radiolabeled polypeptides at the same site within the batch of azide-labeled polypeptides. , and can be obtained by either site-directed modification or random azide conjugation. For example, in the case of random azide conjugation, batch samples of azide-labeled polypeptides containing a single distribution of azide modification sites can be radiolabeled for different purposes using the click chemistry of the invention.

The method of the invention, called "click radiolabeling", which relies on click chemistry, comprises (1) obtaining a first click chemistry reaction partner, e.g., a modified polypeptide, such as an antibody, comprising an azide moiety; ) obtaining a radioactive complex comprising a radioactive metal ion, such as ²²⁵ Ac, ¹¹¹ In, or ⁸⁹ Zr, associated with a chelating moiety, wherein the chelating moiety is a second click chemistry reaction partner; For example, DOTA-dibenzocyclooctyne (DOTA-DBCO) or Deferoxamine-D
(3) a strain-promoted azide-alkyne cycloaddition reaction (SP) between the azide moiety and the alkyne group;
AAC), and conducting a reaction between a click chemistry reaction partner of the modified peptide and a radioactive complex.

The methods of the invention can maximize efficiency by allowing radiometal chelation under low or high pH and/or high temperature conditions, which reduces the risk of inactivating the alkyne reaction partner. can be achieved without Efficient chelation between azide-mAbs and radiocomplexes and efficient SPAAC reactions allow the generation of radioimmunoconjugates with high radiochemical yields even at low azide:mAb ratios. In the methods of the present invention, the only step where trace metals must be excluded is radiometal ion chelation to the chelating moiety, and antibody production, purification, and conjugation steps are performed under metal-free conditions. does not need to be done in

As used herein, the term "click chemistry" refers to the chemical philosophy introduced by Sharpless, which is geared towards rapidly and reliably generating covalent bonds by linking together small units containing reactive groups. explain the proposed chemistry (Kolb, supra,
et al. (see ). Click chemistry does not refer to a specific response, but refers to concepts including, but not limited to, responses that mimic reactions found in nature.
In some embodiments, click chemistry reactions are modular, broad-spectrum, have high chemical yields, produce harmless by-products, are stereospecific, and combine with a single reaction product. It exhibits a large thermodynamic driving force for selecting reactions and/or can be performed under physiological conditions. In some embodiments, click chemistry reactions exhibit high atom economies, can be performed under simple reaction conditions, use readily available starting materials and reagents, and do not use toxic solvents. , or use a non-toxic or easily removed solvent such as water, and/or provide simple product isolation by non-chromatographic methods such as crystallization or distillation. In certain embodiments, the click chemistry reaction is an azide (-
N3) and the Huigen cycloaddition or 1,3-dipolar cycloaddition reaction between an alkyne or alkyne moiety to form a 1,2,4-triazole linker.

In a general aspect, the invention relates to a method of labeling a polypeptide, aptamer or small molecule ligand with a radioactive metal ion, the method comprising:
a. providing a modified polypeptide comprising a polypeptide covalently attached to a first click reaction partner;
b. providing a radioactive complex comprising a radioactive metal ion associated with a chelating moiety;
wherein the chelating moiety comprises a chelating agent covalently attached to the second click reaction partner;
c. contacting the modified polypeptide with a radioactive complex under conditions that allow the first click reaction partner to react with the second click reaction partner to label the polypeptide with a radiometal ion; include.

As used herein, the term "polypeptide" refers to a polymer composed of naturally occurring structural variants and their synthetic non-naturally occurring analogs linked via peptide bonds. The term "polypeptide" refers to a polypeptide of any size, structure, or function. Typically, polypeptides are at least 3 amino acids long. Polypeptides may be naturally occurring, recombinant, or synthetic, or any combination thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. According to preferred embodiments, the polypeptide is an antibody, preferably a monoclonal antibody, or fragment thereof, such as an antigen-binding fragment thereof. According to a preferred embodiment, the antibody or fragment thereof is specific for cancer antigens. According to other embodiments, the polypeptide is a genetically engineered domain or scaffold protein.

As used herein, the term "antibody" or "immunoglobulin" is used in a broad sense and includes immunoglobulin or antibody molecules, including polyclonal antibodies, murine, human, human-adapted, humanized, and chimeric monoclonal antibodies, and Monoclonal antibodies, including antigen-binding fragments thereof, are included.

Antibodies, in general, are proteins or peptide chains that exhibit binding specificity for a particular antigen, and are referred to herein as "targets." The structure of antibodies is known. immunoglobulins are
Five major classes, namely IgA, IgD, depending on the amino acid sequence of the heavy chain constant domain
, IgE, IgG, and IgM. IgA and IgG are further subdivided into the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Thus, antibodies of the invention can be of any of the five major classes or corresponding subclasses. Antibodies of the invention include IgG1, IgG2, IgG3,
or preferably IgG4. The antibody light chains of any vertebrate species can be assigned to one of two distinct types, kappa and lambda, based on the amino acid sequences of their constant domains. Accordingly, an antibody of the invention may contain a kappa or lambda light chain constant domain. According to certain embodiments, the antibodies of the invention comprise heavy and/or light chain constant regions of murine or human antibodies. Each of the four IgG subclasses are
They have distinct biological functions known as effector functions. These effector functions are generally mediated by interaction with Fc receptors (FcγR) or by C1q binding and complement fixation. Binding to FcγRs can lead to antibody-dependent cell-mediated cytolysis, whereas binding to complement factors can lead to complement-mediated cytolysis. Antibodies useful in the present invention may have no or minimal effector functions, but retain their ability to bind FcRn.

As used herein, the term "antigen-binding fragment" includes, for example, diabodies,
Fab, Fab', F(ab')2, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'),
Disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), single domain antibodies (sdab), scFv dimers (bivalent diabodies), one or more CDRs
multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds antigen but does not contain the complete antibody structure Refers to antibody fragments such as An antigen-binding fragment can bind to the same antigen that the parent antibody or parent antibody fragment binds. as used herein
The term "single-chain antibody" refers to a conventional single-chain antibody in the art comprising a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the art comprising a heavy chain variable region and a heavy chain constant region or only a heavy chain variable region. .

As used herein, the term "scaffold" or "scaffold protein"
refers to any protein that has a target binding domain and is capable of binding to a target. A scaffold comprises a mostly structural "framework" and a "binding domain" that contacts a target to provide specific binding. A binding domain of a scaffold need not be defined by one contiguous sequence of the scaffold. In certain cases, the scaffold may be part of a larger binding protein, which itself may be part of a multimeric binding protein comprising multiple scaffolds. A particular binding protein may be bispecific or multispecific, in that it can bind to two or more different epitopes. A scaffold may be derived from a single chain antibody, or a scaffold may not be derived from an antibody.

Any method for chemical or enzymatic modification of polypeptides known to those of skill in the art in view of the present disclosure can be used to covalently attach the polypeptides of the invention to the first click reaction partner. . Amine-reactive groups that react with primary amines present at the N-terminus of each polypeptide chain and the side chains of lysine residues can be used in methods for random modification of polypeptides. Examples of amine-reactive groups suitable for use in the present invention include, but are not limited to, N-hydroxysuccinimide (NHS), substituted NHSs such as sulfo-NHS, isothiocyanates, and tetra- and perfluorophenyl esters. Thiol-reactive groups that react with thiols or sulfhydryls present on the side chains of cysteine residues can be used in methods of random modification of polypeptides. Examples of thiol-reactive groups suitable for use with the present invention include, but are not limited to, maleimide, haloacetyl, and phenyloxadiazole sulfone. According to a preferred embodiment, the modified polypeptide comprises an electrophile covalently attached to a first click reaction partner (eg, NHS-azide);
It is obtained by reacting the side chain of lysine, preferably with an amino side chain.

The methods of the invention further allow the production of site-specifically radiolabeled polypeptides. The click radiolabeling method of the present invention facilitates the site-specific generation of radioimmunoconjugates by utilizing established methods for the site-specific introduction of azide groups into antibodies (Li, X., e.
tal. Preparation of well-defined antibodies
dy-drug conjugates through glycan remode
ling and strain-promoted azide-alkyne cy
loads. Angew Chem Int Ed Engl, 2014
. 53(28):p. 7179-82; Xiao, H.; , et al. , Genetic
Incorporation of multiple unnatural ami
no acids into proteins in mammalian cells
s. Angew Chem Int Ed Engl, 2013.52(52): p. 1
4080-3). Methods for attaching molecules to proteins or antibodies in a site-specific manner are known in the art, and any method for site-specific labeling of antibodies known to those of skill in the art may be used.
can be used in the present invention in view of the present disclosure. Examples of methods for site-specific modification of antibodies suitable for use in the present invention include, but are not limited to, modified cysteine residues (e.g., TH
IOMAB™), unnatural amino acids or glycans (e.g. selenocysteine, p-AcPhe, formylglycine synthase (FGE, SMARTag™)
etc.), and the use of enzymatic methods (eg, glycotransferases, endoglycosidases, microbial or bacterial transglutaminases (MTG or BTG), sortase A, etc.). According to a preferred embodiment, the modified polypeptide comprises the innermost GlcNAc
is left intact in Fc to allow site-specific incorporation of an azido sugar at that site, G
Core Gl of the Fc-glycosylation site of antibodies such as lycINATOR (Genovis)
An antibody or antigen-binding fragment thereof obtained by trimming the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for β-1,4 linkages between cNac residues. The trimmed antibody or antigen-binding fragment thereof is then treated with UDP-N-azidoacetylgalactosamine (UDP-GalN
az) or an azide-labeled sugar such as UDP-6-azido-6-deoxyGalNac to give modified antibodies or antigen-binding fragments thereof. According to another preferred embodiment, the modified polypeptide is an antibody or antigen-binding fragment thereof obtained by glycosylating the antibody or antigen-binding fragment thereof with an amidase. then
The resulting deglycosylated antibody or antigen-binding fragment thereof is treated with an azidoamine, preferably 3-azidopropylamine, 6-azidohexylamine, or any azido-linker amine or any azidoalkylamine, azido-polyethylene glycol (PEG)
-amines and the like, such as O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, O- Modified antibodies or antigen-binding fragments thereof can be obtained by reaction with (2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol or the like, or by reaction in the presence of microbial transglutaminase.

As used herein, the term "aptamer" refers to a single-stranded oligonucleotide (single-stranded DNA or RNA molecule) capable of specifically binding its target with high affinity. Aptamers can be used as molecules to target a variety of organic and inorganic substances.

As used herein, the term "small molecule ligand" refers to low molecular weight organic compounds. As used herein, small molecule ligands can refer to compounds having a size of less than about 1000 Daltons and can be synthesized in the laboratory or found in nature.

As used herein, the term "click reaction partner" or "click chemistry handle" refers to a reactant or reactive group capable of participating in a click chemistry reaction. Click reaction partners are rarely found in naturally occurring biomolecules and are chemically inert to biomolecules, but when reacted with e.g. Reactions can be efficiently carried out under biologically relevant conditions, eg, cell culture conditions such as excess heat or the absence of harsh reactants. In general, a click chemistry reaction requires at least two molecules comprising click reaction partners that are capable of reacting with each other. Such click reaction partners that are reactive with each other are sometimes referred to herein as click chemistry handle pairs, or click chemistry pairs. In some embodiments, the click reaction partner is an azide and a strained alkyne, such as cyclooctyne, or any other alkyne. In other embodiments, the click reaction partner is a reactive diene and a suitable tetrazine dienophile. For example, trans-cyclooctene, norbornene, or biscyclononene can be paired with a tetrazine dienophile suitable as a click reaction partner. In still other embodiments, a tetrazole is paired with an unactivated alkene in the presence of UV light to form a "
A click reaction pair can be created called a "light click" reaction pair. In other embodiments,
Click reaction partners are cysteine and maleimide. For example, a cysteine derived from a peptide (eg GGGC) can be reacted with a maleimide associated with a chelating agent (eg NOTA). Other suitable click chemistry handles are known to those of skill in the art (eg, Spicer et al., Selective ch.
emical protein modification. Nature Commu
nications. 2014; 5:p. 4740). In other embodiments, the click reaction partner is a Staudinger ligation component such as phosphine and azide. In other embodiments, the click reaction partners are dienes such as tetrazines and Diels-Alder reaction components such as alkenes such as trans-cyclooctene (TCO) or norbornene. Exemplary click reaction partners are described in U.S. Patent Application Publication No. 20130266512 and WO2015073746, the relevant descriptions of both click reaction partners being incorporated herein by reference. . According to a preferred embodiment, one of the first and second click reaction partners comprises an alkyne group and the other of the click reaction partners comprises an azide. According to another preferred embodiment, one of the first and second click reaction partners comprises an alkene group and the other of the click reaction partners comprises a diene.

As used herein, the terms "alkyne,""alkynegroup," or "alkyne moiety" refer to a functional group containing a carbon-carbon triple bond. Alkyne moieties include terminal alkynes and cyclic alkynes, preferably terminal and cyclic alkynes reactive with azide groups. A terminal alkyne has at least one hydrogen atom attached to a triple-bonded carbon atom. Cyclic alkynes are cycloalkyl rings containing one or more triple bonds. Examples of cyclic alkynes include, but are not limited to, bicyclononine (BCN), difluorocyclooctyne (DI
FO), dibenzocyclooctyne (DIBO), keto-DIBO, biarylazacyclooctyne (BARAC), dibenzoazacyclooctyne (DIBAC), dimethoxyazacyclooctyne (DIMAC), dibenzocyclooctyne (DBCO), difluorobenzocyclooctyne (DIFBO), monobenzocyclooctyne (MOBO), and tetramethoxyDIBO (TMDIBO) and cyclooctyne and cyclooctyne derivatives. According to a preferred embodiment, one of the first and second click reaction partners comprises a cyclic alkyne, preferably DBCO. According to a preferred embodiment, the other of the click reaction partners comprises an azide, preferably NHS-azide.

As used herein, the term "diene" refers to a compound having two carbon-carbon double bonds and these double bonds attached at the 1,3 positions. The diene double bond can be either cis or trans. Examples of dienes include, but are not limited to, tetrazine or tetrazole groups.

As used herein, the terms "alkene,""alkenegroup," or "alkene moiety" refer to unsaturated hydrocarbon molecules containing carbon-carbon double bonds. According to certain embodiments, alkenes can contain from 2 to 100 carbon atoms. Examples of alkenes include, but are not limited to norbornene and trans-cyclooctene (TCO).
According to another preferred embodiment, one of the first and second click reaction partners comprises an alkene group, preferably norbornene or TCO. According to a preferred embodiment, the other click reaction partner comprises a diene, preferably a tetrazine or tetrazole group.

As used herein, the term "covalently bound" means that the polypeptide is attached to the first click reaction partner through at least one covalent bond and the chelator is attached through at least one covalent bond. Means attached to a second click reaction partner. The binding may be direct, ie without a linker, or indirect, ie through a linker.

As used herein, the term "linker" refers to a chemical moiety that connects a polypeptide or chelator to a click reaction partner. Any suitable linker known to those of skill in the art in light of the present disclosure can be used in the present invention. Linkers are, for example, single covalent bonds, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl moieties, polyethylene glycol (PEG) linkers, peptide linkers, sugar-based linkers, or disulfide bonds;
Or it can be a cleavable linker such as a protease cleavage site such as valine-citrulline-PAB.

As used herein, the term "radioactive metal ion" or "radioactive metal ion"
Refers to one or more isotopes of an element that emit particles and/or photons. Any radiometal known to those of skill in the art in view of the present disclosure can be used in the present invention. Examples of radiometals suitable for use in the present invention include, but are not limited to, ³² P, ⁴⁷ Sc, ⁶² Cu, ⁶⁴ Cu, ⁶⁷
Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁷⁷ As, ⁸⁶ Y, ⁸⁹ Zr, ⁸⁹ Sr, ⁹⁰ Y, ⁹⁹ Tc
, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹¹¹ In, ¹¹⁷ Sn, ¹³¹ I, ¹⁵³ S
m, ¹⁵⁹ Gd, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷⁷ Lu, ¹⁸⁶ Re, ^18
8 Re, ¹⁹⁴ Ir, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹² Pb, ²¹² Bi,
²¹³ Bi, ²²³ Ra, ²²⁵ Ac, ²²⁷ Th, and ²⁵⁵ Fm. As used herein, the term "diagnostic emitter" refers to radioactive metal ions useful in diagnostic or imaging applications. Examples of diagnostic emitters include, but are not limited to, ⁶² Cu, ⁶⁴ Cu,
Gamma emitters such as ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, and ¹¹¹ In are included. As used herein, the term "therapeutic emitter" refers to radioactive metal ions useful in therapeutic applications. Examples of therapeutic emitters include, but are not limited to, beta or alpha emitters such as Thorium, Radium, ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁷⁷ As, ⁸⁹
Sr, ⁹⁰ Y, ⁹⁹ Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹³¹ I, ¹⁵³ Sm
, ¹⁵⁹ Gd, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸
Re, ¹⁹⁴ Ir, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹² Pb, ²¹² Bi, ^2
13 Bi, ²²³ Ra, ²²⁵ Ac, ²⁵⁵ Fm and ²²⁷ Th. According to a preferred embodiment, the radioactive metal ion is ²²⁵ Ac. According to other embodiments, the polypeptide can be labeled with a non-metallic radiolabel for use in pre-targeting or theranostic applications. Examples of non-metallic radiolabels suitable for use in the present invention include, but are not limited to ¹²⁵ I and ¹⁸ F.

The radioactive complexes described herein contain a radioactive metal ion associated with a chelating moiety.
According to embodiments of the present invention, a chelating moiety comprises a chelating agent covalently attached to a click reaction partner, sometimes referred to herein as a "bifunctional chelator."

As used herein, the term "chelating agent" or "chelator" refers to a radioactive metal, such as ²²⁵ Ac, or a chemical compound with which a metal can be chelated via a coordinate bond. Any chelating agent known to those of skill in the art in view of the present disclosure can be used in the present invention.
In one embodiment, the chelating agent comprises a macrocycle. Examples of macrocyclic chelating agents suitable for use in the present invention include, but are not limited to, deferoxamine (DFO), ethylenediaminetetraacetic acid (EDTA), and diethylenetriaminepentaacetic acid (DTPA). In another embodiment, the chelating agent comprises an open chain ligand. Examples of chelating agents containing open chain ligands suitable for use in the present invention include, but are not limited to, 1,4,7,10-tetraazacyclododecane-N,N',N'',N''' - tetraacetic acid (DOTA), 1,4,
7,10,13,16-hexaazacyclohexadecane-N,N',N'',N''',
N'''',N''''-hexaacetic acid (HEHA), 1,4,7,10,13-pentaazacyclopentanadecane-N,N',N'',N''', N''''-pentaacetic acid (P
EPA), Macropa (Thiele et al., An Eighteen-M
embered Macrocyclic Ligand for Actinium-
225 Targeted Alpha Therapy. Angew Chem In
t Ed Engl. 2017 Nov 13;56(46):p. 14712-147
17), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10 - Tetrapropionic acid (DOTPA), 1,4,8,11-tetraazacyclotetradecane-1
,4,8,11-tetrapropionic acid (TETPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid (DOTMP). According to a preferred embodiment, the chelating agent is of formula (I):

の構造
（式中、Ｒ_１、Ｒ_２、Ｒ_３及びＲ_４は、それぞれ独立してＣＨＱＣＯ_２Ｘであり、
Ｑは、独立して、水素、Ｃ_１～Ｃ_４アルキル又は（Ｃ_１～Ｃ_２アルキル）フェニルであ
り、
Ｘは、独立して、水素、ベンジル、Ｃ_１～Ｃ_４アルキルであり、
Ｚは、（ＣＨ２）_ｎＹであり、
ｎは、１～１０であり、
Ｙは、第２のクリック反応パートナーに共有結合した求電子性又は求核性部分であり、
或いは、Ｚは水素である；並びに
Ｒ_１、Ｒ_２、Ｒ_３及びＲ_４は、それぞれ独立してＣＨＱＣＯ_２Ｘであり、
Ｑは、独立して、水素、Ｃ_１～Ｃ_４アルキル又は（Ｃ_１～Ｃ_２アルキル）フェニルであ
り、
Ｘは、独立して、水素、ベンジル、Ｃ_１～Ｃ_４アルキル、又は第２のクリック反応パー
トナーに共有結合した求電子性又は求核性部分である）
を含む。

structure of wherein R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X;
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl,
Z is (CH2) _n Y;
n is 1 to 10,
Y is an electrophilic or nucleophilic moiety covalently attached to the second click reaction partner;
Alternatively, Z is hydrogen; and R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X,
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl, or an electrophilic or nucleophilic moiety covalently attached to a second click reaction partner)
including.

According to a preferred embodiment, the chelating moiety is of formula (II):

の構造を含む。

contains the structure of

According to a preferred embodiment, the chelating moiety is of formula (III):

を含む。

including.

In one embodiment, the invention relates to methods of labeling polypeptides with two or more radioactive metal ions using the methods of the invention. For example, a method of labeling a polypeptide with two radioactive metal ions comprises:
a. providing a modified polypeptide comprising a polypeptide covalently attached to a first click reaction partner and a second click reaction partner;
b. providing a first radioactive complex comprising a first radiometal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently bound to a third click reaction partner;
c. providing a second radioactive complex comprising a second radioactive metal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently bound to a fourth click reaction partner;
d. labeling the polypeptide with first and second radioactive metal ions by reacting the first click reaction partner with a third click reaction partner and the second click reaction partner with a fourth click reaction partner; and contacting the modified polypeptide with the first and second radioactive complexes under conditions that allow for.

According to a preferred embodiment, one of the first and second click reaction partners comprises an alkyne group, the other of the first and second click reaction partners comprises an azide, and the third and fourth
one of the click reaction partners comprises an alkene group and the other of the third and fourth click reaction partners comprises a diene.

According to a preferred embodiment, the first or second radioactive metal ions are diagnostic emitters,
The other is the therapy emitter. According to a preferred implementation, both the first and second radioactive metal ions are therapeutic emitters.

Conditions for conducting click chemistry reactions are known in the art and
Any conditions for conducting click chemistry reactions known to those of skill in the art in view of the present disclosure can be used in the present invention. Examples of conditions include, but are not limited to, incubating the modified polypeptide and radioactive complex at a ratio of 1:1 to 1000:1 at pH 4-10 and temperature 20°C-70°C.

The products of the click radiolabeling method of the invention can be analyzed using methods known to those of skill in the art in view of the present disclosure. For example, LC/MS analysis can be used to determine the ratio of chelator to labeled polypeptide, and analytical size exclusion chromatography can be used to determine the oligomeric state of polypeptides and polypeptide complexes. radiochemical yields can be determined by simple thin layer chromatography (eg iTLC-SG) and radiochemical purity can be measured by size exclusion HPLC. Exemplary methods are described herein, eg, in the Examples below.

Pharmaceutical Compositions and Methods of Treatment The click radiolabeling method of the invention may be modified to a pre-targeting approach (Kra
eber-Bodere, F.; , et al. , A pretargeting sys
tem for tumor PET imaging and radioimmunity
therapy. Front Pharmacol, 2015.6: p. 54). First, the azide-mAb is administered, allowed to bind to target cells, and cleared from circulation over time or removed with a clearing agent. Subsequently, azide-mAbs bound to target sites were administered with radioactive complexes.
Upon undergoing the SPAAC reaction with , the remaining unbound radioactive complex is rapidly cleared from circulation (Deal, KA, et al., Improved in vivo stab
Activity of actinium-225 macrocyclic complex
xes. J Med Chem, 1999.42(15):p. 2988-92). This pre-targeting technique provides a method of enhancing radiometal ion localization at a target site in a subject.

Accordingly, in another general aspect, the invention relates to pharmaceutical compositions comprising a radiolabeled polypeptide prepared by the method of the invention and a pharmaceutically acceptable carrier.

As used herein, the term "carrier" includes any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicles, microspheres, liposome encapsulation. Refers to body or other material known in the art for use in pharmaceutical formulations. It will be appreciated that the nature of the carrier, excipient or diluent will depend on the route of administration for a particular application. The term "pharmaceutically acceptable carrier" as used herein refers to non-toxic materials that do not interfere with the efficacy of the compositions according to the invention or the biological activity of the compositions according to the invention. According to certain embodiments,
Suitable for use in antibody-based or radiocomplex-based pharmaceutical compositions in view of the present disclosure,
Any pharmaceutically acceptable carrier can be used in the present invention.

According to specific embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be administered intravenously, subcutaneously,
It can be formulated so as to be suitable for intramuscular or intratumoral administration.

According to certain embodiments, the modified polypeptide and radioactive complex can be administered in the same or different compositions.

In another general aspect, the invention provides a method of treating a neoplastic disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention. for a method comprising:

According to certain embodiments, the methods of the invention comprise administering a therapeutically effective dose of a pharmaceutical composition of the invention, wherein the composition targets cells associated with the neoplastic disease or disorder. alpha particles from ²²⁵ Ac and its daughter nuclides are delivered to target cells, causing a cytotoxic effect on the target cells and thereby causing a neoplastic disease or disorder. treat.

According to certain embodiments, therapeutically effective amounts of modified polypeptides and radioactive complexes are administered in different compositions.

As used herein, the term "therapeutically effective amount" refers to that amount of active ingredient or component that elicits the desired biological or pharmacological response in a subject. A therapeutically effective amount can be determined empirically and routinely for the stated purposes. For example, in vitro assays can optionally be employed to help identify optimal dosage ranges. Selection of a specific effective amount is based on consideration of several factors, including the disease to be treated or prevented, associated symptoms, patient weight, patient immune status, and other factors known to those of skill in the art. It can be determined by one skilled in the art (eg, by clinical trials). Also, the precise dose to be employed in the formulation will depend on the route of administration, and the severity of the disease, and should be decided according to the judgment of the physician and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

As used herein, the terms "treat", "treating",
and "treatment" are both amelioration of at least one measurable physical parameter associated with a disease, disorder, or condition, such as a neoplastic disease or disorder, for which administration of radioactive metal ions may be beneficial. It is intended to refer to recovery, which is not necessarily perceptible in the subject, but which may be perceptible in the subject. The terms “treat,” “treating,” and “treatment” also refer to causing regression of, preventing progression of, or at least halting the progression of a disease, disorder, or condition. Sometimes it means to delay. In certain embodiments, "treat"
"Treat" and "treatment" refer to one or more diseases, disorders, or conditions in which administration of radioactive metal ions may benefit, such as neoplastic diseases or disorders. Refers to alleviation of symptoms, prevention of progression or onset, or shortening of the period. In specific embodiments, "treat,""treating," and "treatment" refer to preventing recurrence of a disease, disorder, or condition. In specific embodiments, "treat,""treat," and "treatment" refer to improving survival of a subject with a disease, disorder, or condition. In specific embodiments, "treat,""treat," and "treatment" refer to the elimination of a disease, disorder, or condition in a subject.

Examples of neoplastic diseases or disorders include, but are not limited to, disseminated cancer, solid tumor cancer, hypertrophy,
Coronary artery disease or vaso-occlusive disease, diseases or disorders associated with infected cells, microorganisms or viruses, or diseases or disorders associated with inflammatory cells such as rheumatoid arthritis (RA).

The term "subject" as used herein refers to animals, preferably mammals.
According to specific embodiments, the subject is a non-primate (eg, camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig, marmoset, or mouse), or Mammals, including primates (eg, monkeys, chimpanzees, or humans). In a specific embodiment, the subject is human.

Any dosing schedule for modified polypeptides and radioactive complexes can be used in light of the present disclosure. Generally, when the modified polypeptide and the radioactive complex are administered in different compositions, the radioactive complex can be administered at any time after the modified antibody is administered.

According to certain embodiments, compositions used in the treatment of neoplastic diseases or disorders can be used in combination with other agents effective in treating the associated neoplastic disease or disorder.

As used herein, the term "conjointly" refers to the use of multiple therapeutic agents in the context of administration of more than one therapeutic agent to a subject. The use of the term "concurrently" does not limit the order in which the treatments are administered to a subject. For example, a first therapeutic agent (eg, a composition described herein) may be administered to a subject prior to administration of a second therapeutic agent (eg, 5 minutes, 15 minutes, 30 minutes, 4 minutes).
5 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 7 hours
2 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 1
2 weeks before), at the same time, or thereafter (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2
hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after).

In another general aspect, the invention comprises a radiolabeled antibody prepared by the method of the invention and a pharmaceutically acceptable carrier, wherein the immunological properties of the radiolabeled antibody are preserved. , on theranostic agents.

As used herein, the term "theranostic" refers to the ability to provide either diagnostic or therapeutic functions. In one embodiment, theranostic agents provide both diagnostic and therapeutic functions. In another embodiment, the theranostic agent is a non-diagnostic active agent. In yet another embodiment, the theranostic agents are agents that are useful diagnostically but have no therapeutic function.

According to a preferred embodiment, the radioactive metal ions are emitted from a diagnostic emitter, preferably ⁸⁹ Zr
is. According to another preferred embodiment, the radioactive metal ion is a therapeutic emitter, preferably ²²⁵ Ac. According to preferred embodiments, theranostic agents are used to provide both diagnostic and therapeutic functions to subjects in need thereof.

Combinations and Kits Provided herein are combinations comprising:
a. a modified polypeptide comprising a polypeptide covalently attached to a first click reaction partner;
b. a radioactive complex comprising a radioactive metal ion associated with a chelating moiety, the chelating moiety comprising a chelating agent covalently bound to a second click reaction partner,
Here, the combination is used to label the polypeptide with radioactive metal ions.

According to a particular embodiment, the combination of the invention is a reaction mixture used for labeling polypeptides with radioactive metal ions. According to another embodiment, the combination is i
A pack or kit used to produce radiolabeled polypeptides n vitro or in vivo. If desired, the combination may be accompanied by a notice or instructions in the form specified by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice is not applicable for administration to humans. It reflects an agency authorization for manufacture, use, or sale for Use of combinations encompassed herein in methods of labeling the above polypeptides with radioactive metal ions or methods of treating neoplastic diseases or disorders in a subject in need thereof can do.

Embodiments The invention also provides the following non-limiting embodiments.

Embodiment 1 is a method of labeling a polypeptide with a radioactive metal ion, comprising:
a. providing a modified polypeptide comprising a polypeptide covalently attached to a first click reaction partner;
b. providing a radioactive complex comprising a radioactive metal ion associated with a chelating moiety;
wherein the chelating moiety comprises a chelating agent covalently attached to the second click reaction partner;
c. contacting the modified polypeptide with a radioactive complex under conditions that allow the first click reaction partner to react with the second click reaction partner to label the polypeptide with a radiometal ion; include.

Embodiment 1a is the method of embodiment 1, wherein the chelating agent comprises a macrocycle.

Embodiment 1b is the method of embodiment 1, wherein the chelating agent comprises an open chain ligand.

Embodiment 2 is the method of embodiment 1, wherein one of the first and second click reaction partners comprises an alkyne group and the other of the click reaction partners comprises an azide.

Embodiment 3 is the method of embodiment 2, wherein the first click reaction partner comprises an azide group and the second click reaction partner comprises an alkyne group.

Embodiment 3a is a method according to embodiment 2 or 3, wherein the alkyne group comprises a terminal alkyne.

Embodiment 3b is a method according to embodiment 2 or 3, wherein the alkyne group comprises a cyclic alkyne, preferably cyclooctyne or a cyclooctyne derivative.

Embodiment 3c is a method according to embodiment 3b, wherein the alkyne group comprises bicyclononyne (BCN).

Embodiment 3d is a method according to embodiment 3b, wherein the alkyne group comprises difluorinated cyclooctyne (DIFO).

Embodiment 3e is a method according to embodiment 3b, wherein the alkyne group comprises dibenzocyclooctyne (DIBO).

Embodiment 3f is a method according to embodiment 3b, wherein the alkyne group comprises biarylazacyclooctyne (BARAC).

Embodiment 3g is a method according to embodiment 3b, wherein the alkyne group comprises dibenzazacyclooctyne (DIBAC).

Embodiment 3h is wherein the alkyne group comprises dimethoxyazacyclooctyne (DIMAC).
A method according to embodiment 3b.

Embodiment 3i is a method according to embodiment 3b, wherein the alkyne group comprises dibenzocyclooctyne (DBCO).

Embodiment 3j is a method according to embodiment 3b, wherein the alkyne group comprises difluorobenzocyclooctyne (DIFBO).

Embodiment 3k is a method according to embodiment 3b, wherein the alkyne group comprises monobenzocyclooctyne (MOBO).

Embodiment 3l is a method according to embodiment 3b, wherein the alkyne group comprises tetramethoxyDIBO (TMDIBO).

Embodiment 3m is a method according to any one of embodiments 2-3l, wherein the azide group comprises NHS-azido.

Embodiment 4 is the method of embodiment 1, wherein one of the first and second click reaction partners comprises an alkene group and the other of the click reaction partners comprises a diene.

Embodiment 4a is the method of embodiment 4, wherein the diene comprises a tetrazine or tetrazole group.

Embodiment 4b is a method according to Embodiment 4 or 4a, wherein the alkene group comprises norbornene.

Embodiment 4c is a method according to embodiment 4 or 4a, wherein the alkene group comprises trans-cyclooctene (TCO).

Embodiment 5 is a method according to any one of embodiments 1-4c, wherein the polypeptide is an antibody or antigen-binding fragment thereof.

Embodiment 6 is the method of embodiment 5, wherein the antibody is a monoclonal antibody or antigen-binding fragment thereof.

Embodiment 6a is a method according to any one of embodiments 1-6, wherein the modified polypeptide is obtained by randomly conjugating one or more azide groups to the polypeptide.

Embodiment 6b is Embodiments 1-6 wherein the modified polypeptide is a modified antibody or antigen-binding fragment thereof obtained by site-specific incorporation of the first click reaction partner
A method according to any one of

Embodiment 6c provides that the modified antibody, or antigen-binding fragment thereof, is a bacterial endoglycosidase specific for β-1,4 linkages between the core GlcNac residue(s) of the Fc-glycosylation site of the antibody. or trimming an antigen-binding fragment thereof to obtain a trimmed antibody or antigen-binding fragment thereof and a glycosyltransferase, preferably G
A method according to embodiment 6b obtained by reacting the trimmed antibody or antigen binding fragment thereof with an azido sugar, preferably a UDP-GalNaz azido sugar substrate, in the presence of alT galactosyltransferase.

Embodiment 6d comprises the modified antibody or antigen-binding fragment thereof deglycosylating the antibody or antigen-binding fragment thereof with an amidase to obtain the deglycosylated antibody or antigen-binding fragment thereof, and the deglycosylated antibody or antigen-binding fragment thereof. A method according to embodiment 6b, wherein the fragment is obtained by reacting the fragment with an azidoamine, preferably 3-azidopropylamine, in the presence of a microbial transglutaminase.

Embodiment 6e is an antibody that binds to human prostate specific membrane antigen (PSMA) or an antigen-binding fragment thereof, preferably the antibody binds to HC CDR1 of sequence SEQ ID NO:3
Sequence, HC CDR2 sequence of SEQ ID NO:4, HC CDR3 sequence of SEQ ID NO:5, SEQ ID NO:6
light chain (LC) CDR1 sequence of SEQ ID NO:7, LC CDR2 sequence of SEQ ID NO:8, and LC of SEQ ID NO:8
A method according to any one of embodiments 6-6d, comprising the CDR3 sequences.

Embodiment 6f is wherein the antibody comprises the HC sequence of SEQ ID NO:9 and the LC sequence of SEQ ID NO:10.
The method of embodiment 6e.

Embodiment 7 is characterized in that the radioactive metal ion is ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁷⁷ As, ⁸⁹ S
r, ⁹⁰ Y, ⁹⁹ Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹³¹ I, ¹⁵³ Sm,
¹⁵⁹ Gd, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ R
e, ¹⁹⁴ Ir, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹² Pb, ²¹² Bi, ^21
3 Bi, ²²³ Ra, ²²⁵ Ac, ²⁵⁵ Fm, ²²⁷ Th, ⁶² Cu, ⁶⁴ Cu, ⁶⁷
The method of any one of embodiments 1-6d, wherein Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, or ¹¹¹ In.

Embodiment 7a is the method of any one of embodiments 1-6d, wherein the radioactive metal ion is ²²⁵ Ac.

Embodiment 7b is the method of any one of embodiments 1-6d, wherein the radioactive metal ion is ¹¹¹ In.

Embodiment 7c is any one of Embodiments 1-6d wherein the radioactive metal ion is ⁸⁹ Zr.
The method described in 1.

Embodiment 8 is the method of any one of embodiments 1-7c, wherein the chelating moiety is covalently attached to the second click reaction partner via a linker.

Embodiment 9 reacts a sulfhydryl group covalently attached to a first click reaction partner with an electrophile on a side chain, preferably the amino side chain of a lysine on or introduced into the polypeptide. 9. The method of any one of embodiments 1-8, further comprising obtaining a modified polypeptide, preferably NHS-azide.

Embodiment 10 is a method according to any one of embodiments 1-9, wherein the modified polypeptide comprises a polypeptide covalently attached, either directly or through a linker, to an azide, tetrazine, or tetrazole group.

Embodiment 11 provides that the chelating agent is a macrocycle, preferably of formula (I):

の構造
（式中、Ｒ_１、Ｒ_２、Ｒ_３及びＲ_４は、それぞれ独立してＣＨＱＣＯ_２Ｘであり、
Ｑは、独立して、水素、Ｃ_１～Ｃ_４アルキル又は（Ｃ_１～Ｃ_２アルキル）フェニルであ
り、
Ｘは、独立して、水素、ベンジル、Ｃ_１～Ｃ_４アルキルであり、
Ｚは、（ＣＨ２）_ｎＹであり、
ｎは、１～１０であり、
Ｙは、第２のクリック反応パートナーに共有結合した求電子性又は求核性部分であり、
或いは、Ｚは水素である；並びに
Ｒ_１、Ｒ_２、Ｒ_３及びＲ_４は、それぞれ独立してＣＨＱＣＯ_２Ｘであり、
Ｑは、独立して、水素、Ｃ_１～Ｃ_４アルキル又は（Ｃ_１～Ｃ_２アルキル）フェニルであ
り、
Ｘは、独立して、水素、ベンジル、Ｃ_１～Ｃ_４アルキル、又は第２のクリック反応パー
トナーに共有結合した求電子性又は求核性部分である）
を含む、実施形態１～１０のいずれか１つに記載の方法である。

structure of wherein R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X;
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl,
Z is (CH2) _n Y;
n is 1 to 10,
Y is an electrophilic or nucleophilic moiety covalently attached to the second click reaction partner;
Alternatively, Z is hydrogen; and R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X,
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl, or an electrophilic or nucleophilic moiety covalently attached to a second click reaction partner)
11. The method of any one of embodiments 1-10, comprising

Embodiment 12 provides that the chelating moiety has formula (II):

の構造を含む、実施形態１～１１のいずれか１つに記載の方法である。

12. The method of any one of embodiments 1-11, comprising the structure of

Embodiment 12a is where the chelating moiety comprises a chelating agent having an open chain ligand, preferably the chelating moiety is of formula (III):

の構造を含む、実施形態１～１１のいずれか１つに記載の方法である。

12. The method of any one of embodiments 1-11, comprising the structure of

Embodiment 12b provides that the chelating moiety is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), deferoxamine (DFO), 1
,4,7,10,13,16-hexaazacyclohexadecane-N,N′,N″,N′
'',N'''',N''''-hexaacetic acid (HEHA), 1,4,7,10,13-
Pentaazacyclopentanadecane-N,N',N'',N''',N''''-pentaacetic acid (PEPA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (
DTPA), Macropa (Thiele et al., An Eighteen-
Membered Macrocyclic Ligand for Actinium
-225 Targeted Alpha Therapy. Angew Chem I
nt Ed Engl. 2017 Nov 13;56(46):p. 14712-14
717), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10 -Tetrapropionic acid (DOTPA), 1,4,8,11-tetraazacyclotetradecane-
selected from the group consisting of 1,4,8,11-tetrapropionic acid (TETPA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid (DOTMP); 11. The method of any one of embodiments 1-10, wherein the chelating agent comprises:

Embodiment 12c is in which the chelating agent is 1,4,7,10-tetraazacyclododecane-N
, N′,N″,N′″-tetraacetic acid (DOTA).

Embodiment 12d is Embodiment 12b wherein the chelating agent comprises deferoxamine (DFO)
is the method described in

Embodiment 13 is a radiometal ion, preferably ²²⁵ Ac, ¹¹¹ In or ⁸⁹ Zr
A method of labeling a polypeptide, preferably an antibody or antigen-binding fragment thereof, using
a. providing a modified polypeptide, preferably a modified antibody or antigen-binding fragment thereof, comprising a polypeptide covalently attached to an azide, tetrazine, or tetrazole group, or an antibody or antigen-binding fragment thereof;
b. ²²⁵ Ac, ¹¹¹ In associated with a radioactive metal ion, preferably a chelating moiety
or ⁸⁹ Zr, wherein the chelating moiety comprises a chelating agent covalently attached to an alkyne or alkene group; and c. The azide, tetrazine, or tetrazole group reacts with an alkyne or alkene group to convert the polypeptide or antibody or antigen-binding fragment thereof to a radioactive metal ion,
contacting the modified polypeptide or antibody or antigen-binding fragment thereof with a radioactive complex, preferably under conditions that permit labeling with ²²⁵ Ac, ¹¹¹ In, or ⁸⁹ Zr;
The chelating agent has the formula (I):

（式中、Ｒ_１、Ｒ_２、Ｒ_３及びＲ_４は、それぞれ独立してＣＨＱＣＯ_２Ｘであり、
Ｑは、独立して、水素、Ｃ_１～Ｃ_４アルキル又は（Ｃ_１～Ｃ_２アルキル）フェニルであ
り、
Ｘは、独立して、水素、ベンジル、Ｃ_１～Ｃ_４アルキルであり、
Ｚは、（ＣＨ２）_ｎＹであり、
ｎは、１～１０であり、
Ｙは、アルキン基に共有結合した求電子性又は求核性部分であり、
或いは、Ｚは水素である；並びに
Ｒ_１、Ｒ_２、Ｒ_３及びＲ_４は、それぞれ独立してＣＨＱＣＯ_２Ｘであり、
Ｑは、独立して、水素、Ｃ_１～Ｃ_４アルキル又は（Ｃ_１～Ｃ_２アルキル）フェニルであ
り、
Ｘは、独立して、水素、ベンジル、Ｃ_１～Ｃ_４アルキル、又はアルキン基に共有結合し
た求電子性又は求核性部分である）
の構造を含む、方法である。

(wherein R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X,
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl,
Z is (CH2) _n Y;
n is 1 to 10,
Y is an electrophilic or nucleophilic moiety covalently attached to the alkyne group;
Alternatively, Z is hydrogen; and R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X,
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently an electrophilic or nucleophilic moiety covalently bonded to a hydrogen, benzyl, C ₁ -C ₄ alkyl, or alkyne group)
A method comprising the structure of

Embodiment 13a provides that the chelating agent is 1,4,7,10-tetraazacyclododecane-N
,N',N'',N'''-tetraacetic acid (DOTA).

Embodiment 13b is a chelating moiety of formula (II):

の構造を含む、実施形態１３に記載の方法である。

14. The method of embodiment 13, comprising the structure of

Embodiment 13d is a radiometal ion, preferably ²²⁵ Ac, ¹¹¹ In or ⁸⁹ Z
A method of labeling a polypeptide, preferably an antibody or antigen-binding fragment thereof, with r, comprising:
a. providing a modified polypeptide, preferably a modified antibody or antigen-binding fragment thereof, comprising a polypeptide covalently attached to an azide, tetrazine, or tetrazole group, or an antibody or antigen-binding fragment thereof;
b. ²²⁵ Ac, ¹¹¹ In associated with a radioactive metal ion, preferably a chelating moiety
or ⁸⁹ Zr, wherein the chelating moiety comprises a chelating agent covalently attached to an alkyne or alkene group; and c. The azide, tetrazine, or tetrazole group reacts with an alkyne or alkene group to convert the polypeptide or antibody or antigen-binding fragment thereof to a radioactive metal ion,
contacting the modified polypeptide or antibody or antigen-binding fragment thereof with a radioactive complex, preferably under conditions that permit labeling with ²²⁵ Ac, ¹¹¹ In, or ⁸⁹ Zr;
Here is the method wherein the chelating agent comprises an open chain ligand, preferably deferoxamine (DFO).

Embodiment 14 is a method according to any one of embodiments 13-13C, wherein the chelating agent is covalently attached to the alkyne or alkene group via a linker.

Embodiment 15 comprises a sulfhydryl group covalently attached to an azide, preferably NHS-azide, and an electrophile on a side chain, preferably on a polypeptide or a polypeptide preferably
reacting the amino side chain of lysine introduced into the antibody or antigen-binding fragment thereof,
further comprising obtaining a modified polypeptide, or an antibody or antigen-binding fragment thereof;
15. The method of any one of embodiments 13-14.

Embodiment 16 is a method according to any one of embodiments 13-15, wherein the polypeptide, preferably an antibody or antigen-binding fragment thereof, is covalently attached to the azide via a linker.

Embodiment 17 provides that the polypeptide is an antibody or antigen-binding fragment thereof, the radioactive metal ion is ²²⁵ Ac, ¹¹¹ In or ⁸⁹ Zr, and the chelating moiety is of formula (II
):

の構造を含む、実施形態１３に記載の方法である。

14. The method of embodiment 13, comprising the structure of

Embodiment 17a provides that the polypeptide is an antibody or antigen-binding fragment thereof, the radioactive metal ion is ²²⁵ Ac, ¹¹¹ In or ⁸⁹ Zr, and the chelating moiety is of formula (I
II):

の構造を含む、実施形態１３ｃに記載の方法である。

13c. The method of embodiment 13c comprising the structure of

Embodiment 17b is an antibody wherein the polypeptide binds to human prostate specific membrane antigen (PSMA) or an antigen-binding fragment thereof, preferably the antibody is HC of sequence SEQ ID NO:3
CDR1 sequence, HC CDR2 sequence of SEQ ID NO:4, HC CDR3 sequence of SEQ ID NO:5,
The method of any one of embodiments 13-17a, comprising the light chain (LC) CDR1 sequence of SEQ ID NO:6, the LC CDR2 sequence of SEQ ID NO:7, and the LC CDR3 sequence of SEQ ID NO:8.

Embodiment 17c is a method according to embodiment 17b, wherein the antibody comprises the HC sequence of SEQ ID NO:9 and the LC sequence of SEQ ID NO:10.

Embodiment 18 is a method of dual labeling a polypeptide with two radioactive metal ions, comprising:
a. providing a modified polypeptide comprising a polypeptide covalently attached to a first click reaction partner and a second click reaction partner;
b. providing a first radioactive complex comprising a first radiometal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently bound to a third click reaction partner;
c. providing a second radioactive complex comprising a second radioactive metal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently bound to a fourth click reaction partner;
d. labeling the polypeptide with first and second radioactive metal ions by reacting the first click reaction partner with a third click reaction partner and the second click reaction partner with a fourth click reaction partner; and contacting the modified polypeptide with the first and second radioactive complexes under conditions that allow for.

Embodiment 19 is wherein one of the first and second click reaction partners comprises an alkyne group,
wherein the other of the first and second click reaction partners comprises an azide, one of the third and fourth click reaction partners comprises an alkene group, and the other of the third and fourth click reaction partners comprises a diene. 19. The method of embodiment 18.

Embodiment 20 is a method according to embodiment 18 or 19, wherein the first or second radioactive metal ions are diagnostic emitters and the other is a therapeutic emitter.

Embodiment 21 is a method according to embodiment 18 or 19, wherein both the first and second radioactive metal ions are therapeutic emitters.

Embodiment 21a has diagnostic emitters comprising: ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶
22. The method of embodiment 20 or 21, wherein Y, ⁸⁹ Zr, or ¹¹¹ In.

Embodiment 21b is in which the therapeutic emitter comprises ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁷⁷ As, ⁸⁹ S
r, ⁹⁰ Y, ⁹⁹ Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹³¹ I, ¹⁵³ Sm,
¹⁵⁹ Gd, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ R
e, ¹⁹⁴ Ir, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹² Pb, ²¹² Bi, ²¹
Embodiment 20- which is ³ Bi, ²²³ Ra, ²²⁵ Ac, ²⁵⁵ Fm, or ²²⁷ Th
21a.

Embodiment 22 is a pharmaceutical composition comprising a radiolabeled polypeptide prepared by the method of any one of embodiments 1-21b and a pharmaceutically acceptable carrier.

Embodiment 23 is a method of treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder, in a subject in need of treatment or diagnosis of the disease or disorder, particularly a neoplastic disease or disorder, comprising: 22. A method comprising administering the composition of 22 to a subject.

Embodiment 24 is the embodiment wherein the pharmaceutical composition comprises two compositions administered sequentially, the first composition comprising the modified polypeptide and the second comprising the radioactive complex(s). 23
is the method described in

Embodiment 25 comprises a radiolabeled antibody prepared by the method of any one of embodiments 1-21b and a pharmaceutically acceptable carrier, wherein the immunological properties of the radiolabeled antibody are preserved. It is a theranostic agent.

Embodiment 26 is the theranostic agent according to embodiment 25, wherein the radioactive metal ion is a diagnostic emitter, preferably ^89Zr .

Embodiment 27 is wherein the radioactive metal ion is a therapeutic emitter, preferably ²²⁵ Ac.
A theranostic agent according to embodiment 25.

Embodiment 27a is a theranostic agent according to embodiment 25, wherein the radioactive metal ion is ¹¹¹ In.

Embodiment 27b is an antibody wherein the polypeptide binds to human prostate specific membrane antigen (PSMA) or an antigen-binding fragment thereof, preferably the antibody is HC of sequence SEQ ID NO:3
CDR1 sequence, HC CDR2 sequence of SEQ ID NO:4, HC CDR3 sequence of SEQ ID NO:5,
The theranostic agent according to any one of embodiments 25-27a, comprising the light chain (LC) CDR1 sequence of SEQ ID NO:6, the LC CDR2 sequence of SEQ ID NO:7, and the LC CDR3 sequence of SEQ ID NO:8.

Embodiment 27c is a method according to embodiment 27b, wherein the theranostic agent comprises the HC sequence of SEQ ID NO:9 and the LC sequence of SEQ ID NO:10.

Embodiment 27d provides that the radiolabeled antibody has formula (IV):

式（ＩＶ）（ＤＯＴＡ－Ａｃ－ＤＢＣＯ－タンパク質）の式を有するか、或いは、^２２
５Ａｃが、^３２Ｐ、^４７Ｓｃ、^６７Ｃｕ、^７７Ａｓ、^８９Ｓｒ、^９０Ｙ、^９９Ｔｃ、^１０
５Ｒｈ、^１０９Ｐｄ、^１１１Ａｇ、^１３１Ｉ、^１５３Ｓｍ、^１５９Ｇｄ、^１６５Ｄｙ、^１
６６Ｈｏ、^１６９Ｅｒ、^１７７Ｌｕ、^１８６Ｒｅ、^１８８Ｒｅ、^１９４Ｉｒ、^１９８Ａｕ
、^１９９Ａｕ、^２１１Ａｔ、^２１２Ｐｂ、^２１２Ｂｉ、^２１３Ｂｉ、^２２３Ｒａ、^２５５
Ｆｍ、^２２７Ｔｈ、^６２Ｃｕ、^６４Ｃｕ、^６７Ｇａ、^６８Ｇａ、^８６Ｙ、^８９Ｚｒ、又は
^１１１Ｉｎなどの別の放射性金属イオンで置換されている、実施形態２５～２７ｃのいず
れか１つに記載のセラノスティック剤である。

having the formula (IV) (DOTA-Ac-DBCO-protein) ^;
5 Ac is ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁷⁷ As, ⁸⁹ Sr, ⁹⁰ Y, ⁹⁹ Tc, ^10
5 Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹³¹ I, ¹⁵³ Sm, ¹⁵⁹ Gd, ¹⁶⁵ Dy, ^1
66 Ho, ¹⁶⁹ Er, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁴ Ir, ¹⁹⁸ Au
, ¹⁹⁹ Au, ²¹¹ At, ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²³ Ra, ²⁵⁵
Fm, ²²⁷ Th, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, or
The theranostic agent according to any one of embodiments 25-27c substituted with another radioactive metal ion, such as ¹¹¹ In.

Embodiment 27e provides that the radiolabeled antibody has formula (V):

式（Ｖ）（ＤＯＴＡ－Ｉｎ－ＤＢＣＯタンパク質）
の式を有する、実施形態２５～２７ｃのいずれか１つに記載のセラノスティック剤であ
る。

Formula (V) (DOTA-In-DBCO protein)
The theranostic agent according to any one of embodiments 25-27c, having the formula:

Embodiment 27f provides that the radiolabeled antibody has formula (VI):

式（ＶＩ）（ＤＦＯ－Ｚｒ ＤＢＣＯ－タンパク質）の式を有するか、或いは、^８９Ｚ
ｒが、^３２Ｐ、^４７Ｓｃ、^６７Ｃｕ、^７７Ａｓ、^８９Ｓｒ、^９０Ｙ、^９９Ｔｃ、^１０５Ｒ
ｈ、^１０９Ｐｄ、^１１１Ａｇ、^１３１Ｉ、^１５３Ｓｍ、^１５９Ｇｄ、^１６５Ｄｙ、^１６６
Ｈｏ、^１６９Ｅｒ、^１７７Ｌｕ、^１８６Ｒｅ、^１８８Ｒｅ、^１９４Ｉｒ、^１９８Ａｕ、^１
９９Ａｕ、^２１１Ａｔ、^２１２Ｐｂ、^２１２Ｂｉ、^２１３Ｂｉ、^２２３Ｒａ、^２２５Ａｃ
、^２５５Ｆｍ、^２２７Ｔｈ、^６２Ｃｕ、^６４Ｃｕ、^６７Ｇａ、^６８Ｇａ、^８６Ｙ、又は^１
１１Ｉｎなどの別の放射性金属イオンで置換されている、実施形態２５～２７ｃのいずれ
か１つに記載のセラノスティック剤である。

having the formula (VI) (DFO-Zr DBCO-protein); or ⁸⁹ Z
r is ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁷⁷ As, ⁸⁹ Sr, ⁹⁰ Y, ⁹⁹ Tc, ¹⁰⁵ R
h, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹³¹ I, ¹⁵³ Sm, ¹⁵⁹ Gd, ¹⁶⁵ Dy, ¹⁶⁶
Ho, ¹⁶⁹ Er, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁴ Ir, ¹⁹⁸ Au, ^1
99 Au, ²¹¹ At, ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²³ Ra, ²²⁵ Ac
, ²⁵⁵ Fm, ²²⁷ Th, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, or ¹
The theranostic agent according to any one of embodiments 25-27c substituted with another radioactive metal ion, such as ¹¹ In.

Embodiment 27g provides that the radiolabeled antibody has formula (VII):

式（ＶＩＩ）（ＤＯＴＡ－Ｚｒ－ＤＢＣＯ－タンパク質）
の式を有する、実施形態２５～２７ｃのいずれか１つに記載のセラノスティック剤であ
る。

Formula (VII) (DOTA-Zr-DBCO-protein)
The theranostic agent according to any one of embodiments 25-27c, having the formula:

Embodiment 27h is wherein the radiolabeled antibody has a chelator:antibody ratio (CAR) of less than 3.
A theranostic agent according to any one of embodiments 25-27g.

Embodiment 27i is the theranostic agent according to any one of embodiments 25-27h, wherein the radiolabeled antibody has a chelator:antibody ratio (CAR) of two.

Embodiment 28 is
a. a modified polypeptide comprising a polypeptide covalently attached to a first click reaction partner;
b. a radioactive complex comprising a radioactive metal ion associated with a chelating moiety, the chelating moiety comprising a chelating agent covalently bound to a second click reaction partner,
Here, the combination is used to label the polypeptide with radioactive metal ions.

Embodiment 28a is the combination or kit of embodiment 28, wherein the chelating agent comprises a macrocycle.

Embodiment 28b is the combination according to embodiment 28, wherein the chelating agent comprises an open chain ligand.

Embodiment 29 is used to label a polypeptide with radiometal ions in vitro via a reaction between first and second click reaction partners, Embodiment 28
A combination or kit according to .

Embodiment 30 is a combination or kit according to embodiment 28 used for labeling a polypeptide with radiometal ions in vivo via a reaction between first and second click reaction partners .

Embodiment 31 is a composition comprising a modified polypeptide comprising a polypeptide covalently attached to a first click reaction partner.

Embodiment 32 is a composition comprising a radioactive complex comprising a radiometal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently bound to a second click reaction partner. .

Embodiment 32a is a composition according to embodiment 32, wherein the chelating agent comprises a macrocycle.

Embodiment 32b is a composition according to embodiment 32, wherein the chelating agent comprises an open chain ligand.

Embodiment 33 is a combination or kit according to any one of embodiments 28-30, or a composition according to embodiment 31 or 32, wherein the polypeptide is an antibody or antigen-binding fragment thereof.

Embodiment 33a provides that the antibody is capable of binding human prostate specific membrane antigen (PSMA) or an antigen-binding fragment thereof, preferably the antibody comprises the HC CDRs of sequence SEQ ID NO:3
1 sequence, the HC CDR2 sequence of SEQ ID NO:4, the HC CDR3 sequence of SEQ ID NO:5, the light chain (LC) CDR1 sequence of SEQ ID NO:6, the LC CDR2 sequence of SEQ ID NO:7, and the L of SEQ ID NO:8.
34. The method of embodiment 33, comprising a C CDR3 sequence.

Embodiment 33b is a combination or kit or composition according to embodiment 33a, wherein the antibody comprises the HC sequence of SEQ ID NO:9 and the LC sequence of SEQ ID NO:10.

Embodiment 33c is a combination or kit or composition according to embodiments 33a or 33b, wherein the antibody or antigen-binding fragment thereof is covalently attached to an azide group.

Embodiment 33d is the combination or kit or composition of embodiment 33c, wherein the antibody or antigen-binding fragment thereof is randomly covalently attached to the azide groups.

Embodiment 33e is the combination or kit or composition of embodiment 33c, wherein the antibody or antigen-binding fragment thereof is site-specifically covalently attached to an azide group.

Embodiment 33f provides that the antibody or antigen-binding fragment thereof is a bacterial endoglycosidase specific for β-1,4 linkages between the core GlcNac residue(s) of the Fc-glycosylation site of the antibody or trimming the antigen-binding fragment thereof to obtain a trimmed antibody or antigen-binding fragment thereof and a glycosyltransferase, preferably Ga
covalently attached to the azide group via a method comprising reacting the trimmed antibody or antigen-binding fragment thereof with an azido sugar, preferably a UDP-GalNaz azido sugar substrate, in the presence of lT galactosyltransferase. 33e or a kit or composition.

Embodiment 33g provides that the modified antibody or antigen-binding fragment thereof deglycosylate the antibody or antigen-binding fragment thereof with an amidase to obtain the deglycosylated antibody or antigen-binding fragment thereof, and the deglycosylated antibody or antigen-binding fragment thereof. A combination or kit or composition according to embodiment 33e, obtained by a method comprising reacting the fragment with an azidoamine, preferably 3-azidopropylamine, in the presence of a microbial transglutaminase.

Embodiment 34 is wherein the radioactive metal ion comprises ²²⁵ Ac, ¹¹¹ In, or ⁸⁹ Zr.
A combination, kit or composition according to any one of embodiments 33-33g.

Embodiment 35 provides that the chelating agent is a macrocycle, preferably of formula (I):

の構造
（式中、Ｒ_１、Ｒ_２、Ｒ_３及びＲ_４は、それぞれ独立してＣＨＱＣＯ_２Ｘであり、
Ｑは、独立して、水素、Ｃ_１～Ｃ_４アルキル又は（Ｃ_１～Ｃ_２アルキル）フェニルであ
り、
Ｘは、独立して、水素、ベンジル、Ｃ_１～Ｃ_４アルキルであり、
Ｚは、（ＣＨ２）_ｎＹであり、
ｎは、１～１０であり、
Ｙは、アルキン基に共有結合した求電子性又は求核性部分であり、
或いは、Ｚは水素である；並びに
Ｒ_１、Ｒ_２、Ｒ_３及びＲ_４は、それぞれ独立してＣＨＱＣＯ_２Ｘであり、
Ｑは、独立して、水素、Ｃ_１～Ｃ_４アルキル又は（Ｃ_１～Ｃ_２アルキル）フェニルであ
り、
Ｘは、独立して、水素、ベンジル、Ｃ_１～Ｃ_４アルキル、又はアルキン基に共有結合し
た求電子性又は求核性部分である）
を含む、実施形態３４に記載の組み合わせ、キット又は組成物である。

structure of wherein R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X;
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl,
Z is (CH2) _n Y;
n is 1 to 10,
Y is an electrophilic or nucleophilic moiety covalently attached to the alkyne group;
Alternatively, Z is hydrogen; and R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X,
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently an electrophilic or nucleophilic moiety covalently bonded to a hydrogen, benzyl, C ₁ -C ₄ alkyl, or alkyne group)
35. A combination, kit or composition according to embodiment 34 comprising

Embodiment 36 is a combination, kit or composition according to embodiment 35, wherein the electrophilic or nucleophilic moiety is covalently attached to the alkyne group via a linker.

Embodiment 37 provides that the chelating moiety has formula (II):

の構造を含む、実施形態３５又は３６に記載の組み合わせ、キット又は組成物である。

37. A combination, kit or composition according to embodiment 35 or 36 comprising the structure of

Embodiment 37a is wherein the chelating moiety comprises a chelating agent having an open-chain ligand, preferably the chelating moiety is of formula (III):

の構造を含む、実施形態３４に記載の組み合わせ、キット又は組成物である。

35. A combination, kit or composition according to embodiment 34 comprising the structure of

Embodiment 38 is a combination, kit or composition according to any one of embodiments 34-37, wherein the polypeptide is covalently attached to the azide via a linker.

Embodiment 39 is a method of treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder, comprising a theranostic agent according to any one of embodiments 25-27d, or embodiments 28 and 33- 39. A method comprising administering a combination according to any one of 38 to a subject.

Embodiment 40 is a method of treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder, in a subject in need of treatment or diagnosis of a disease or disorder, particularly a neoplastic disease or disorder, wherein embodiment 31 and the composition of embodiment 32 to a subject, preferably wherein the polypeptide is an antibody.

The following examples of the invention are intended to further illustrate the nature of the invention. It should be understood that the following examples do not limit the invention, the scope of the invention being defined by the appended claims.

Example 1: Random Conjugation of Azide/Handle to Antibody Monoclonal Antibody (mAb): Designated as "PSMB127", herein referred to as "anti-PS
Human IgG4 that binds to human prostate specific membrane antigen (PSMA), termed "MA mAb"
The antibody has the heavy chain (HC) CDR1 sequence of SEQ ID NO:3, the HC CDR2 sequence of SEQ ID NO:4, the HC CDR3 sequence of SEQ ID NO:5, the light chain (LC) CDR1 sequence of SEQ ID NO:6, the LC of SEQ ID NO:7 It has a CDR2 sequence, and an LC CDR3 sequence of SEQ ID NO:8, and an HC sequence of SEQ ID NO:9 and an LC sequence of SEQ ID NO:10. Anti-PSMA mAbs were expressed and purified using standard chromatographic methods.

Human IgG4 S228P, an anti-PMSA mAb referred to herein as the "control mAb"
The /F234A/L235A (IgG4-PAA) antibody isotype control has the HC sequence of SEQ ID NO:1 and the LC sequence of SEQ ID NO:2. Commercially available antibodies trastuzumab (Herceptin), cetuximab (Erbitux), pertuzumab (Perjeta), and panitumumab (Vectibix) were obtained from Roche, Lilly, Roche, and Amgen, respectively.
purchased from Mouse anti-human Her2 mAb was obtained from BioXCell (catalog number BE02).
77). Trastuzumab, Pertuzumab, and anti-human Her2 mAbs bind to human Her2. Cetuximab and panitumumab bind to human EGFR.

Conjugation: Stock solution of antibody (1-10 mg/mL) in 10 mM sodium acetate pH 5.2, phosphate buffered saline pH 7, or other compatible buffer at 20% (w/w) of 1
A final pH of about 9 was obtained by mixing with M sodium carbonate buffer pH 9. NHS-PEG
4-Azide (Thermo Catalog No. 26130) was dissolved in DMSO to a final concentration of 1
00 mM and a 0.2% (wt/wt) stock solution was added to generate a molar excess of approximately 3-10 over the mAb. After incubating the reaction for 10 min at 22° C., 1M Tris
(pH 7.5) was added to give a final concentration of 50 mM Tris.

Purification: Zeba desalting column with 7K MW cutoff (Thermo), dialysis,
The azide-mAb conjugate was purified using a method such as standard protein A affinity chromatography, or another compatible method, and dissolved in a compatibility buffer (PBS; 20 mM HEP
ES 150 mM, NaCl pH 7.5; or 10 mM sodium acetate pH 5.2). After purification, Amico with 50K MW cutoff (Millipore)
The conjugate was concentrated to 10-20 mg/mL using an n-concentrator.

LC/MS Analysis: The chelator:antibody ratio (CAR) was measured on an Agilent PLRP-S column (300-Angstrom, 2.1 x 150 mm, catalog number PL1912-33).
01) by LC/MS analysis using an Agilent G6224 MS-TOF instrument (Table 1). Mass spectra were analyzed in m/
Deconvolution was performed using the maximum entropy algorithm over the z-range 2000-3200.

Analytical Size Exclusion Chromatography (SEC): Analytical SEC was performed to determine the oligomeric state of the antibodies and antibody conjugates and to confirm that the conjugation process did not result in aggregation. Tosoh TSKgel G3000SWxl (Tosoh Bioscience
ce #08541) An Agilent 1200 series HPLC with a 7.8 mm x 30 cm column was used. Mobile phase 1×PBS, flow rate 0.8 ml/ml, injection volume: 15 μ
L, protein concentration 0.1-2 mg/mL.

Example 2: Random Conjugation of Azide/Handles to Non-Antibody Polypeptides
systems (catalog number 2914-HT) and dissolved in water at 10 mg/mL
made it EGF, human epidermal growth factor (EGF), was isolated from Sino Biological
(catalog number 10605-HNAE).

Conjugation: stock solution of polypeptide (1-10 mg/mL) in 10 mM sodium acetate pH 5.2, phosphate buffered saline pH 7, or other compatible buffer, 20% (w/w) 1M sodium carbonate Mixed with buffer pH 9 to a final pH of about 9. NHS-
PEG4-Azide (Thermo Catalog No. 26130) was dissolved in DMSO to a final concentration of 100 mM and a 0.2% (wt/wt) stock solution was added to produce approximately a 3-10 molar excess over protein. bottom. After incubating the reaction at 22° C. for 10 min,
Final concentration was 50 mM while quenching with Tris pH 7.5. Conjugation efficiency is shown in Table 2.

Example 3: Site-Specific Incorporation of Azidosugars into Antibody Glycans Antibody glycans are combined with bacterial endoglycosylation specific for β-1,4 linkages between core GlcNac residues within the Fc glycosylation site(s). GlycINATOR, a glycosidase
(Genovis), leaving the innermost GlcNac intact on the Fc, which can later be used for site-specific incorporation of azido sugars. More specifically, GlycINATOR immobilized on agarose beads packed in a column (Genovis)
was equilibrated in Tris-buffered saline pH 7.4 (TBS). 5-10 mg/mL
of mAb was added to the resin, incubated on a rocker for 1 hour at room temperature, and eluted by spinning at 100×g for 1 minute. The column was eluted with 0.5 mL TBS three times. Eluates containing trimmed mAb were pooled and buffer additives supplied (Genovis) were added along with UDP-GALNaz azidosugar substrate and GalT galactosyltransferase enzyme. The reaction mixture was stirred overnight at 30°C. The final azide mAb was purified using a mAb selection column (GE) on an AKTA Avant instrument. Azide modification was confirmed by LC-MS and determined to have a CAR of exactly 2.

Example 4 Site-Specific Incorporation of 3-Azidopropylamine by Microbial Transglutaminase (MTG) The azide group was site-specifically placed on the antibody essentially at position Gln295, as described (Dennler et al. .Transglutaminase-based
chemo-enzymatic conjugation approach yi
elds homogeneous antibody-drug conjugate
s. Bioconj Chem 2014 Mar 19;25(3):p. 569-7
8). The anti-PSMA mAb cleaves between the innermost GlcNac residue and the asparagine residues of high-mannose, hybrid and complex oligosaccharides and is conserved (e.g.
Fc Asn297) and non-conserved (e.g., Fab N-glycans) are amidases that allow complete deglycosylation and release of all N-glycans, including N-glycans derived from both glycosylation sites. , Rapid PNGase F (New
(England Biolabs). sodium acetate buffer (
10 mL of 1 mg/mL antibody with 5 μL of PNGase F in pH 5.2).
°C overnight and deglycosylation was confirmed by LC-MS. PNGase
F was removed by 4 cycles of concentration and diluted with an Amicon device (50 KDa cutoff). For conjugation of 3-azidopropylamine (3-APA; Click Chemistry Tools), deglycosylated mAb (0.5-1 mg /mL) was brought to pH 7-7.5. 100
An equivalent amount of 3-APA was added to the MTG activin TI transglutaminase (Ajino
moto) added with 5-10% weight/volume. After incubating the reaction at 37° C. for 1-4 hours, the azide-modified mAb was purified on a mAbSelect Sure column using standard chromatographic methods. The conjugate was hand characterized by LC-MS and showed a CAR of 2
determined to be

Example 5: Chelation of Radiometals to Bifunctional Chelators (BFCs)
Synthesis of ²²⁵ Ac-DOTA-Ga-DBCO: ²²⁵ Ac(NO ₃ ) ₃ was converted to Oak R
It was purchased from idge National Laboratory. 1,4,7,10-
Tetraazacyclododecane, 1-(glutaric acid)-4,7,10-triacetic acid-(3-amino-propanoic acid) dibenzocyclo-octyne (DOTA-Ga-DBCO) was custom synthesized. Synthesis is according to Bernhard et al. Chem. Eur. J. 2012, 18
, 7834-7841. DBCO-amine (3-amino-1-[(5-aza-
3,4:7,8-dibenzocyclooct-1-yn)-5-yl]-1-propanone (S
igma) was reacted with DOTA-GA anhydride and the product was purified by reverse phase HPLC.

Quantification of actinium-225 was accomplished using a Capintec CRC-55TW dose calibrator, whereby ²²⁵ Ac(NO ₃ ) ₃ was dissolved in 0.1N HCl to make a 10 mCi/mL solution. Tetramethylammonium acetate (1 M solution, 7.5 μL, 7.5 μmol) in a plastic vial, DOTA-GA-DBCO (
1 mg/mL aqueous solution, 2.5 μL, 3.4 nmol) and NaOH (0.1 N, 2.5 μL)
L, 0.25 μmol) to a solution of ²²⁵ Ac(NO ₃ ) ₃ (0.1 N HCl 5 μL
medium 10 mCi/mL, 50 μCi, 0.0038 nmol) was added. The pH of the mixture was observed to be approximately 6.5 by pH paper. The vial was placed on a shaking block at 80° C. and 290 rpm for 30 minutes to cool the vial to room temperature.

Synthesis of ¹¹¹ In-DOTA-GA-DBCO: ¹¹¹ InC in 0.05 M HCl
13 was purchased from GE Healthcare. Tetramethylammonium acetate (1 M solution, 7.5 μL, 7.5 μmol) in a plastic vial, DOTA-GA-DB
CO (1 mg/mL aqueous solution, 2.5 μL, 3.4 nmol) and HCl (0.1 N, 5 μL)
L) of ¹¹¹ InCl ₃ in 0.05N HCl (5 μL, Capintec
104.3 μCi, 0.0022 nm as measured by CRC-55TW dose calibrator
ol) was added. The pH of the mixture was observed to be approximately 5.5 by pH paper. The vials were placed on a shaker block at 60° C. and 290 rpm for 30 minutes to cool the vials to room temperature.

Synthesis of ^89Zr -DFO-DBCO: ^89Zr oxalate was purchased from 3D Imaging. DFO-DBCO was purchased from Macrocyclics (Plano, TX, catalog number B-773), dissolved in DMSO to 0.5 mg/mL and diluted to 25% with water.
Diluted to μg/mL.

2 mCi of Zr-89 was transferred to a metal-free microcentrifuge tube and 1 M oxalic acid was added to bring the total volume to 80 μL. 12 μL of 2M potassium carbonate was added in 2 μL portions and the mixture was stirred with a pipette tip until bubbling stopped. Then 120 μL of 1M HE
PES was added followed by 300 μL of water. Test the pH of the solution and adjust p if necessary
Additional 2M potassium carbonate was added to raise the H to 6-6.5. 136 μL of DFO-DBCO stock (3.4 μg) was added and the reaction was incubated for 1 hour at room temperature. 0.5 μL of reactions were analyzed by spotting onto TLC Green strips (Biodex) and eluting with 20% NaCl. After elution, a PerkinElmer Cyclon
The strips were scanned with an e Plus phosphor imager to show chelation by DFO-DBCO.

Synthesis of ⁸⁹ Zr-DOTA-GA-DBCO: Water, Sep-pak Light Q
MA strong anion exchange cartridge (acrylic acid/acrylamide copolymer on diol silica, surface sensitivity: C(O)NH(CH ₂ ) ₃ N(CH ₃ ) ₃ ⁺ Cl ⁻ , pore size 300 Å
, particle size 37-55 μm, ion exchange capacity 230 μeq/gram), MeCN (6 mL)
was added, followed by 0.9% saline (10 mL) and then water (10 mL). 1
. A solution of ⁸⁹ Zr(ox) ₂ in 0 M oxalic acid (2 μL, 290 μCi) was added to the preconditioned cartridge. The cartridge was then washed with deionized water (20 ml).
to remove excess oxalic acid, followed by 1.0 M HCl (aq) (100 μL each).
^89ZrCl4 was eluted from the column with a recovery of 248 μCi (86%) in a _total volume of 400
μl was obtained and most of the radioactivity was in fraction 3. The combined organic extracts were then evaporated to dryness.

10 μL DOTA-GA-DBCO (1.0 mg/mL metal-free water, 10 μL
g, 13.6 nmol) was added to ⁸⁹ ZrCl ₄ (268 uCi, 50 μL). DO
The TA-GA-DBCO/ ⁸⁹ Zr solution was diluted with 150 μL of 1.0 M HEPES. The pH of the mixture was adjusted to pH 7.5 (Pandya et al., Zirconi
um tetraazamacrocycle complexes display
extraordinary stability and provide a ne
w strategy for zirconium-89-based radiop
pharmaceutical development. Chem Sci. 2017
Mar 1;8(3):p. 2309-2314). The solution was then incubated at 90°C for 60 minutes. The yield of ⁸⁹ Zr-DOTA-GA-DBCO complex is 1% NH ₄ OH
Solution eluted SPC25 column (Sigma Aldrich part number SPC251
20-50G) to 98%. The unchelated ⁸⁹ Zr remains on the column while ^{the 89} Zr-DOTA-GA-DBCO complex is eluted.

Example 6 Synthesis of Click-Labeled Radioconjugates of Anti-PSMA mAbs See FIGS. 1 and 2 for schematic representations of radiolabeled antibodies according to the methods of the present invention.

Anti-PSMA mAb- in PBS or other compatible buffer (10-20 mg/mL)
Dibenzo-[1,2,3]-triazoloazocine-Ga-DOTA-225Ac (site-directed, CAR=2): random or site-directed azide-modified antibody (site-directed, CAR=2
or random, average CAR from 1 to 4), ²²⁵ Ac-DO produced as described.
Added to the solution of TA-GA-DBCO. The final pH of the mixture was approximately 6.5 by pH paper. The reaction solution was gently stirred and added to 15 mL of NaOAc buffer, 10 mM, pH 6.
˜6.5, or left at room temperature for 3 hours before purification on a PD-10 column (GE Healthcare) preconditioned with another compatible buffer. The reaction mixture was pipetted into the reservoir of the preconditioned PD-10 column and the eluate was collected in a plastic tube. The reaction vial was washed with NaOAc buffer (0.2m
3×L), the wash solution was pipetted into the reservoir of the Pd-10 column and the eluate collected. NaOAc buffer was applied continuously to the reservoir of the PD-10 column and the eluate was collected in plastic tubes, collecting approximately 1 mL of eluate in each tube until a total of 10 mL of eluate was collected.

Purity of each fraction collected was assessed by iTLC-SG (Agilent) using citrate-H ₂ O-MeOH solution as mobile phase. pure fraction(s)
were combined to give the final product in 10 mM NaOAc buffer. Product solutions were analyzed by HPLC for chemical and radiochemical purity. Antibody concentrations in product solutions were measured by UV absorption using a standard curve. Then Capintec CRC-5
A 5TW dose calibrator was used to quantify the radioactivity of the product solutions.

AC-225 Chelation Quality Control: A chelation challenge using diethylenetriaminepentaacetic acid (DTPA) was used as a quality control for the purified product. 10 mM _Na5
The DTPA aqueous solution is added to the sample solution containing the ²²⁵ Ac-labeled mAb [DPT
A]/[mAb]=500-1,000. 50 mM Na ₅ DTPA aqueous solution, ²
[ ^DPTA
]/[mAb]=50,000 to 100,000. The two mixtures were
Placed on a shake block for 30 minutes at 0 rpm. The mixture was spotted on iTLC-SG,
Citrate-H ₂ O-MeOH was developed as mobile phase. Under these conditions, free ²²⁵
Ac migrates to the solvent front and bound ²²⁵ Ac-mAb remains at baseline.

Anti-PSMA mAb-dibenzo-[1,2,3]-triazoloazocine-GA-DOT
A- Synthesis of ¹¹¹ In: Random or site-specific azide-modified anti-PSMA mAbs (site-specific, CAR = 2 or random, average CAR of 1-4) in 10 mM NaOAc were generated as described for ¹¹¹ Ac. -DOTA-GA-DBCO solution. The reaction solution was gently stirred and allowed to stand at room temperature for 2 hours before passing through a PD-10 column.
A PD-10 column was loaded with 15 mL of NaOAc buffer, 10 mM, pH 6.
Precondition by passing through ~6.5 and discard wash. The reaction mixture was then pipetted into the reservoir of the preconditioned PD-10 column and the eluate was collected in a plastic tube. The reaction vial was washed with NaOAc buffer (0.
2 mL x 3), the wash solution was pipetted into the reservoir of the PD-10 column, and the eluate was collected. NaOAc buffer was applied continuously to the reservoir of the PD-10 column and the eluate was collected in plastic tubes, collecting approximately 1 mL of eluate in each tube until a total of 10 mL of eluate was collected.

Purity of each fraction collected was assessed by iTLC-SG using 10 mM EDTA aqueous solution (pH=5-6) as mobile phase. Combine the pure fraction(s) and
The final product was obtained in 10 mM NaOAc buffer. Product solutions were analyzed by HPLC for chemical and radiochemical purity. Antibody concentrations in product solutions were determined by UV absorption using a standard curve. The radioactivity of the product solutions was then quantified using a Capintec CRC-55TW dose calibrator.

Quality control of In-111 chelation: A chelation challenge using DTPA as a quality control of the purified product was used: 10 mM Na ₅ DTPA in water was added to the sample solution containing ¹¹¹ In-labeled mAb. , [DTPA] / [mAb] = 1000 ~
10,000. The mixture was placed on a shaking block for 30 minutes at room temperature and 290 rpm. The mixture was spotted on iTLC-SG and washed with 10 mM EDTA aqueous solution (pH=5-6).
) was developed as the mobile phase. Free ¹¹¹ In or loosely bound ¹¹¹ migrates to the solvent front, while tightly bound ¹¹¹ In-mAb remains at baseline.

Anti-PSMA mAb-dibenzo-[1,2,3]-triazoloazocine-DFO-ZR
Synthesis of -89 Random azide-modified anti-PSMA mAbs in 10 mM HEPES 50 mM NaCl pH 7.5, or other compatible buffer (average CAR of 1-4, similar procedure can be performed with site-specific azide-mAbs ) was added to a ⁸⁹ Zr-DFO-DBCO solution prepared as described and incubated at 37° C. for 1.5 hours before passing through a PD-10 column. A PD-10 column was passed through the column with 15 mL of isotonic saline and the wash was discarded. The reaction mixture was then pipetted into the reservoir of the preconditioned PD-10 column and the eluate was collected in a plastic tube. Saline was continuously applied to the reservoir of the PD-10 column and the eluate was collected in plastic tubes, collecting approximately 0.5 mL of eluate in each tube until a total of 10 mL of eluate was collected. The radioactivity of each fraction and the material retained on the column was measured with a dose calibrator. The product peak, usually fractions 4-7, were pooled to give the final product. Product solutions were analyzed by HPLC for chemical and radiochemical purity. Antibody concentrations in product solutions were determined by UV absorption using a standard curve. A dose calibrator was used to quantify the radioactivity of the product solutions.

Synthesis of anti-PMSA mAb-DOTA- ⁸⁹ Zr 20 mM HEPES 50 mM NaCl pH 7.5 (10.1 mg/mL, 200 µ
Azide-modified anti-PS modified by random azide conjugation in L, ~13.5 nmol)
MA mAb conjugates (CAR=1-4) were generated as described above with ⁸⁹ Zr-DOTA-
Added to the solution of GA-DBCO. The final pH of the mixture was adjusted to 7.0. The reaction solution is 3
Incubate at 7° C. for 2 hours, followed by purification on a PD-10 column (GE Healthcare) in either 0.9% saline, HEPES buffer, or PBS to obtain 6
The product ⁸⁹ Zr-DOTA-mAb was obtained in 4%.

Quality control of Zr-89 chelation: For Zr-DFO-mAbs, purified products were analyzed by HPLC only. These conjugates did not retain Zr-89 when challenged with DTPA or EDTA. Zr-DOTA-mAbs were challenged by adding EDTA to 33 mM and incubated overnight at room temperature. Post-challenge conjugates were analyzed by passage over a PD-10 column and found to retain 80% of the radioactivity.

Example 7: Analytical Characterization of Click-Labeled Radioconjugates Determination of Radiochemical Conversion Radiochemical conversion (% RA conversion; see Tables 3-5) was measured using iTLC-SG (silica gel (SG) impregnated binder). determined by simplified thin layer chromatography (iTLC) using non-glass microfiber chromatography paper. The %RA conversion value is
Divide the integral of the radiological signal of the product peak (at different retention times of the radioactive starting material and by-products) by the value obtained when integrating all radiological signal peaks present between the baseline and the solvent front is calculated by The percentage of this product is then expressed as conversion.

For products incorporating Ac-225, a sample solution containing approximately 0.1-1 μCi of ²²⁵ Ac was spotted onto the baseline of the iTLC-SG strip approximately 2 cm from the bottom edge. iTLC-SG strips were immersed in a citric acid-water-methanol mobile phase (20 mL 0
. 4M trisodium citrate/3 mL 2N HCl/2.3 mL MeOH), dried at room temperature and stored for a minimum of 6 hours prior to analysis (225Ac and all daughter nuclides reach secular equilibrium). iTLC-SGs were scanned using a Bioscan AR2000 radiographic TLC imaging scanner using 99mTc settings.

For the In-111 chelate, the baseline of the iTLC-SG strip approximately 2 cm from the lower edge was spotted with sample solution containing approximately 0.5-2.5 μCi of ¹¹¹ In. The iTLC-SG strips were developed using 10 mM EDTA pH 5-6 as mobile phase and then dried at room temperature. Bioscan A using the In-111 setting
The dried iTLC-SG was scanned using an R2000 radiographic TLC imaging scanner.

For Zr-89 chelates, %RA conversion was determined by dividing the radioactivity for the product peak by the total radioactivity including the radioactivity on the PD-10 column, as determined by counting on the dose calibrator.

Determining Radiochemical Purity of Radiolabeled Proteins Radiochemical purity (% RA purity, see Tables 3-4) of Ac-225 and In-111 chelates was determined by SE-HPLC (size exclusion HPLC). . Ac-22
For 5, a Tosoh TSKgel column (G3000SW×17.8 mm×30
cm, 5 μm) and the column was eluted with DPBS buffer (×1, calcium and magnesium free). Flow rate: 0.7 mL/min, 20 min run, room temperature. HPLC
Afterwards, the eluate was collected into pre-numbered vials, collecting either 0.5 min or 1 min eluate fractions in each vial. Vials containing the eluate were left at room temperature for more than 6 hours to allow ²²⁵ Ac to reach secular equilibrium with its daughter nuclide. The radioactivity in each vial was then
Counted on a pintec CRC-55TW well counter. A radiochromatogram was reconstructed based on the radioactivity in the vial.

For In-111, a Tosoh TSKgel column (G3000SW×17.
8 mm×30 cm, 5 μm) was used. The column was eluted with DPBS buffer (x1, without calcium and magnesium). Flow rate: 0.7 mL/min, run for 20 minutes,
room temperature. HPLC system and Perkin Elmer radioactive flow detector Rad
Radioactivity detection was performed using an iomatic 625TR, Ultima FloTM using a 0.5 mL flow cell using the In-111 setting and a flow rate of 1.4 mL/min.
It has an M cocktail.

For Zr-89 (see Table 5), Tosoh TSKgel columns (G
3000 SW×17.8 mm×30 cm, 5 μm) was used. The column was eluted with citrate buffered saline. Flow rate: 1 mL/min, 20 minutes run, room temperature. Radioactivity was detected using the HPLC system described above and a Beckman flow-through detector coupled to a Bioscan Flow Count instrument.

^＊工程１は、アクチニウム－２２５のＤＯＴＡ－ＧＡ－ＤＢＣＯへのキレート化である
。
^＊＊工程２は、タンパク質に対する二官能性キレートのクリック反応である。

^* Step 1 is the chelation of actinium-225 to DOTA-GA-DBCO.
^** Step 2 is the click reaction of the bifunctional chelate to the protein.

^＊工程１は、インジウム－１１１のＤＯＴＡ－ＧＡ－ＤＢＣＯへのキレート化である。
^＊＊工程２は、タンパク質に対する二官能性キレートのクリック反応である。

^* Step 1 is the chelation of Indium-111 to DOTA-GA-DBCO.
^** Step 2 is the click reaction of the bifunctional chelate to the protein.

Example 8: Click reaction of modified mAbs with DOTA-GA-DBCO Random and site-specific azide-mAbs (anti-PSMA mAb,
cetuximab, panitumumab, trastuzumab, pertuzumab) were mixed with 5- to 20-fold excess of unchelated DOTA-GA-DBCO and incubated at room temperature or 37°C for 1-24 hours. The mAb was desalted with Zeba desalting dehydration columns (Thermo) and A
Concentrated in a micon centrifuge (Millipore), rediluted with buffer, and concentrated again to remove any remaining DBCO-DOTA. DBCO-DO of all free azides
Complete click reaction with TA was confirmed by LC-MS.

Example 9: Click Reaction of Modified mAbs with DFO-DBCO
Mixed with 5- to 20-fold excess of unchelated DOTA-GA-DFO and incubated at room temperature or 37°C for 1-24 hours. The mAb was applied to a Zeba desalting dehydration column (The
rmo), concentrated in an Amicon centrifuge (Millipore), rediluted with buffer and concentrated again to remove any remaining DBCO-DFO. Complete click reaction of all free azide with DBCO-DFO was confirmed by LC-MS.

Example 10: Cell Binding (FACS)
The cell binding of the azide modified antibody, the DOTA-DBCO-azido modified antibody and the DFO-DBCO-azido modified antibody were compared to the parent mAbs of the conjugates listed in Table 1. Cell lines expressing mAb targets were treated with a range of concentrations of antibody or conjugate and binding was measured by flow cytometry. Panitumumab and cetuximab conjugates were evaluated for binding to EGFR+ A431 cells. Herceptin and pertuzumab were evaluated for binding to HER2+ SK-BR-3 cells. Anti-PSMA mAbs were evaluated for binding to PSMA+C4-2b cells.

Cell lines: C4-2B cells, a human prostate cancer cell line purchased from Janssen Oncology (
(Springhouse, Pennsylvania). A431, a human epidermoid carcinoma cell line
and SK-BR-3 cells, a human breast cancer cell line, Janssen BioTherapeutics using cells originally from ATCC (Manassas, VA).
(Springhouse, PA). EGFR receptor-negative MOLM-1
3 Human acute myeloid leukemia suspension cells were maintained in RPMI 1640+25 mM Hepes (Gibco) supplemented with 20% heat-inactivated fetal bovine serum (Gibco). cells, 10
RPMI 1640 + 25 m with % FBS (Gibo, Waltham, Mass.)
Propagation was performed using MHEPES (Gibo, Waltham, Mass.).

Flow cytometry: Cells were detached from flasks using enzyme-free cell dissociation buffer (Gibco, Waltham, MA) and filtered through a 40 μm filter (Fa
lcon). 5×10 ⁴ cells were seeded per well in 96-well U-bottom plates. Cells were incubated with conjugated or parental antibodies diluted in BSA staining buffer (BD Bioscience, San Jose, CA) for 1 hour at 4°C. Cells were washed twice with staining buffer. Cells were then incubated in the dark for 30 minutes at 4°C.
AlexaFluor647-tagged anti-human IgG secondary antibody (Jackson Immu
no Research Laboratories). Secondary antibodies were diluted 1:200 in staining buffer containing 3% donkey serum (Rockland Immunochemicals). In the final 10 min incubation, S
YTOX™ Green Nucleic Acid Stain (ThermoF
isher) was added to the cells at a final concentration of 30 nM. Cells were washed twice with staining buffer, then resuspended in a final volume of 25 μL/well in staining buffer and placed on the iQue Screen.
ner flow cytometer (Intellicyt). ForeCyt software was used to analyze the data. Viable cells were determined by excluding events with high nucleic acid staining. Mean fluorescence intensity (MFI) was determined in live cells and expressed with GraphPad Prism 7 (G
GraphPad Software). A non-linear regression curve fit was applied to the data and _EC50 values were calculated.

For all mAbs and conjugates tested, parental and modified mAbs showed similar cell binding (Table 6).

Example 11 In-111 Cell Binding Assay Cell binding was measured in a radiometric assay using the In-111 radiolabeled proteins listed in Table 4. Anti-PSMA mAb and transferrin were injected into C4-2B cells (PSMA
+ and transferrin +). Cetuximab and panitumumab were tested against A431 cells (EGFR+) and MOLM-13 cells (EGFR-).

Adherent cells were detached with enzyme-free cell dissociation buffer (Gibco). Detached adherent cells and recovered floating cells were counted and treated with cold staining buffer (BD Biosciences).
). Various numbers of cells in 200 μL staining buffer were added to microcentrifuge tubes and placed on ice. Add 0.5 μCi of In-111 labeled protein to each tube and place on ice for 1
incubated for hours. Cells were washed with cold PBS (Gibco) to remove unbound antibody and resuspended in 500 μL cold PBS. Samples were transferred to counting vials and cell-associated radioactivity was measured by gamma counting (Hidex Automatic Gamma Counter).

The Counts per minute (CPM) from the test sample was transferred to a known amount of In−
Using CPM values with a linear regression made using 111-labeled proteins, In-1
converted to 11 μCi. The µCi boundary value was calculated using the following calculations to determine the molar binding (Moles
Bound): (μCi bound/specific activity)/MW mAb or protein.
Each data point is the mean±SD of triplicate reactions.

Click-labeled In-111 anti-PSMA mAb and In-111 transferrin
It bound to C4-2B cells and increased cell-associated radioactivity with increasing cell number (Fig. 3A). Click-labeled In-111 anti-EGFR antibodies panitumumab and cetuximab
It bound to cells, and cell-associated radioactivity increased with increasing cell number. Negative control MOLM-13
No specific binding of the click-labeled anti-EGFR antibody was detected in the cells (Fig. 3B).

Example 12: Indium Cellular Uptake Assay The kinetics of internalization of In-111 click-labeled anti-PSMA mAb in C4-2B cells was determined.

Cells were seeded at 3×10 ⁶ cells per 60 mm dish (Corning) and 37
Place overnight in a humidified _CO2 incubator at °C. Seeding medium was removed and replaced with 2 mL of cold staining buffer (BD Bioscience). The dish was then placed on ice. 0.5 μCi of In-111 labeled antibody was added to each dish and incubated on ice for 1 hour. Cells were washed with cold PBS (Gibco) to remove unbound antibody from the cell surface. At various time points, cells were assayed for surface membrane-bound and intracellular radioactivity as described below.

Acid wash stripping procedure was used to strip surface bound radioactivity: 1.5 mL
of stripping buffer (50 mm glycine, 150 mm NaCl pH 2.7,
Pepsin (Amresco) added to 25 μg/mL) was added to the cells and the dishes were incubated on ice for 15 minutes. Stripping buffer was transferred to counting vials. Cells were washed with cold PBS and the wash was transferred to counting vials. Radioactivity was assayed by gamma counting (Hidex Automatic Gamma Counter). Surface membrane-bound radioactivity was determined as the sum of stripping buffer plus PBS washes.

Intracellular radioactivity was assayed by preparing cell lysates: after stripping surface-bound radioactivity and washing the cells, 1.5 mL of 1 M NaOH (Teknova) was added to the cells and the dishes were placed on ice for 5 minutes. incubated. surface strip cell lysate,
Transferred to counting vials. The dishes were washed with cold PBS and the wash was transferred to counting vials. Radioactivity was assayed by gamma counting (Hidex Automatic Gamma Counter). Intracellular radioactivity was determined as the sum of surface strip cell lysates plus PBS washes.

For time=0 samples, cells were assayed for surface membrane-bound and intracellular radioactivity immediately after initial antibody binding on ice. For the 10 min, 30 min, 1 hr and 2 hr samples, 3 mL of cell culture medium was added to each dish after initial antibody binding and the dishes were placed in a 37° C. humidified CO ₂ incubator. At each time point the dish was removed from the incubator and placed on ice. Cell culture media was transferred to counting vials and cells were washed with cold PBS. The PBS wash was collected in counting vials. Cells were assayed for surface membrane-bound and intracellular radioactivity as described. At each time point, an unstripped sample was made by incubating the cells with PBS instead of stripping buffer prior to cell lysis. These samples were used to evaluate the stripping efficiency and the results compared to the stripped samples.

CPM from test samples were converted to μCi In-111 using CPM values from a linear regression made using known amounts of In-111 labeled mAb. The percent localization of In-111 mAb on the cell surface membrane (stripped sample) and intracellularly (lysed sample) was calculated using the following formula: percent localization=100 ^* (sample μCi/average total μCi), where total μCi refers to the sum of all collected samples including incubation medium, PBS wash, glycine rinse, and lysed cells. Each data point is the mean±SD of triplicate reactions.

Surface-bound In-111 was rapidly cleared from the cell surface and redistributed intracellularly. The stripping technique releases approximately 80% of the cell surface associated radioactivity at time 0 and by the end of the incubation only 20% is released by stripping and over 60% of the radioactivity is in the cell lysate. There was (Fig. 4).

Example 13: Efficacy in a Mouse Tumor Xenograft Model Anti-PSMA mAb-DOTA-AC-225 Dosimetry Study: Male NSG Mice (1
n=8 per group) were subcutaneously implanted with 10 ⁶ LNCap cells and tumors were allowed to grow to 100-150 mm ³ . Mice were given a range of radioactivity (10 nCi, 25 nCi, 70 nCi, 200 nC
A single dose of i) anti-PSMA mAb-azido-DOTA- ²²⁵ Ac or isotype control, control mAb-azido-DOTA- ²²⁵ Ac was injected intravenously. The injection volume per mouse was brought to a total of 10 μg protein with cold antibody. Tumor measurements and body weights were recorded twice weekly. Animals were euthanized when tumor size exceeded 1,500 mm ³ or when body weight loss exceeded 20%.

The anti-PSMA mAb-DOTA-Ac-225 shows tumor growth inhibition especially after a single dose of high radiation doses and also shows tumor growth inhibition superior to isotype control at all doses (
Figure 5A). Control mAb radioconjugates at both doses had survival curves similar to vehicle controls (Fig. 5B; Table 7); The response was shown (Fig. 5C; Table 7). After completing the test after 209 days,
All three mice remained from the anti-PSMA mAb200nCi group and showed no detectable tumors.

The embodiments of the invention are intended to be exemplary only, and those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures of the invention. could be All such equivalents are considered to be within the scope of this invention and are covered by the following claims.

All references (including patent applications, patents or publications) cited in this specification are by reference in their entirety to indicate that each individual publication or patent or patent application is incorporated in its entirety for any purpose. incorporated herein for all purposes to the same extent as if specifically and individually indicated to be incorporated by reference.

（式中、Ｒ _１ 、Ｒ _２ 、Ｒ _３ 及びＲ _４ は、それぞれ独立してＣＨＱＣＯ _２ Ｘであり、
Ｑは、独立して、水素、Ｃ _１ ～Ｃ _４ アルキル又は（Ｃ _１ ～Ｃ _２ アルキル）フェニルであり、
Ｘは、独立して、水素、ベンジル、Ｃ _１ ～Ｃ _４ アルキルであり、
Ｚは、（ＣＨ２） _ｎ Ｙであり、
ｎは、１～１０であり、
Ｙは、前記第２のクリック反応パートナーに共有結合した求電子性又は求核性部分であり、
或いは、Ｚは水素である；並びに
Ｒ _１ 、Ｒ _２ 、Ｒ _３ 及びＲ _４ は、それぞれ独立してＣＨＱＣＯ _２ Ｘであり、
Ｑは、独立して、水素、Ｃ _１ ～Ｃ _４ アルキル又は（Ｃ _１ ～Ｃ _２ アルキル）フェニルであり、
Ｘは、独立して、水素、ベンジル、Ｃ _１ ～Ｃ _４ アルキル、又は前記第２のクリック反応パートナーに共有結合した求電子性又は求核性部分である）の構造を有するマクロサイクルを含む、
又は、前記キレート剤が、開鎖配位子を含む、上記［１］に記載の方法。
［１５］ 前記キレート化部分が、式（ＩＩ）：

の構造、又は式（ＩＩＩ）：

の構造を含む、上記［１］に記載の方法。
［１６］ ^２２５ Ａ _Ｃ 、 ^１１１ Ｉｎ、又は ^８９ Ｚｒで抗体又はその抗原結合フラグメントを標識する方法であって、
ａ．アジド、テトラジン、又はテトラゾール基に共有結合した抗体又はその抗原結合フラグメントを含む修飾抗体又はその抗原結合フラグメントを提供することと、
ｂ．キレート化部分に会合した ^２２５ Ａ _Ｃ 、 ^１１１ Ｉｎ又は ^８９ Ｚｒを含む放射性錯体を提供することと、ここで、前記キレート化部分が、アルキン又はアルケン基に共有結合したキレート剤を含み、並びに
ｃ．前記アジド、テトラジン、又はテトラゾール基が前記アルキン又はアルケン基と反応することによって、前記抗体又はその抗原結合フラグメントを ^２２５ Ａ _Ｃ 、 ^１１１ Ｉｎ、又は ^８９ Ｚｒで標識化することを可能にする条件下で、前記修飾抗体又はその抗原結合フラグメントを放射性錯体と接触させること、を含み、
前記キレート剤が、式（Ｉ）：

（式中、Ｒ _１ 、Ｒ _２ 、Ｒ _３ 及びＲ _４ は、それぞれ独立してＣＨＱＣＯ _２ Ｘであり、
Ｑは、独立して、水素、Ｃ _１ ～Ｃ _４ アルキル又は（Ｃ _１ ～Ｃ _２ アルキル）フェニルであり、
Ｘは、独立して、水素、ベンジル、Ｃ _１ ～Ｃ _４ アルキルであり、
Ｚは、（ＣＨ２） _ｎ Ｙであり、
ｎは、１～１０であり、
Ｙは、前記アルキン基に共有結合した求電子性又は求核性部分であり、
或いは、Ｚは水素である；並びに
Ｒ _１ 、Ｒ _２ 、Ｒ _３ 及びＲ _４ は、それぞれ独立してＣＨＱＣＯ _２ Ｘであり、
Ｑは、独立して、水素、Ｃ _１ ～Ｃ _４ アルキル又は（Ｃ _１ ～Ｃ _２ アルキル）フェニルであり、
Ｘは、独立して、水素、ベンジル、Ｃ _１ ～Ｃ _４ アルキル、又は前記アルキン基に共有結合した求電子性又は求核性部分である）の構造を含む、
又は、前記キレート剤は、開鎖配位子を含む、方法。
［１７］ 側鎖上の求電子物質を前記アジド、テトラジン又はテトラゾール基に共有結合したスルフヒドリル基と反応させて、前記修飾抗体又はその抗原結合フラグメントを得ることを更に含む、上記［１６］に記載の方法。
［１８］ 前記修飾抗体又はその抗原結合フラグメントが、前記第１のクリック反応パートナーの部位特異的組み込みによって得られる、上記［１６］に記載の方法。
［１９］ 前記修飾抗体又はその抗原結合フラグメントが、前記抗体のＦｃ－グリコシル化部位のコアＧｌｃＮａｃ残基間のβ－１，４結合に特異的な細菌エンドグリコシダーゼで抗体又はその抗原結合フラグメントをトリミングして、トリミングされた抗体又はその抗原結合フラグメントを得ること、及び糖転移酵素の存在下で、前記トリミングされた抗体又はその抗原結合フラグメントをアジド標識糖と反応させることにより、修飾抗体又はその抗原結合フラグメントを得ること、を含む方法によって得られる、上記［１８］に記載の方法。
［２０］ 前記アジド標識糖が、ＵＤＰ－Ｎ－アジドアセチルガラクトサミン（ＵＤＰ－ＧａｌＮａｚ）又はＵＤＰ－６－アジド６－デオキシＧａｌＮＡｃである、上記［１９］に記載の方法。
［２１］ 前記糖転移酵素が、ＧａｌＴガラクトシルトランスフェラーゼ又はＧａｌＮＡｃトランスフェラーゼから選択される、上記［１９］に記載の方法。
［２２］ 前記修飾抗体又はその抗原結合フラグメントが、抗体又はその抗原結合フラグメントをアミダーゼで脱グリコシル化して、脱グリコシル化抗体又はその抗原結合フラグメントを得ること、及び前記脱グリコシル化抗体又はその抗原結合フラグメントを、微生物トランスグルタミナーゼの存在下でアジドアミンと反応させることによって、前記修飾ポリペプチドを得ること、を含む方法によって得られる、上記［１６］に記載の方法。
［２３］ 前記アジドアミンが、３－アジドプロピルアミン、６－アジドヘキシルアミン、Ｏ－（２－アミノエチル）－Ｏ’－（２－アジドエチル）テトラエチレングリコール、Ｏ－（２－アミノエチル）－Ｏ’－（２－アジドエチル）ペンタエチレングリコール、及びＯ－（２－アミノエチル）－Ｏ’－（２－アジドエチル）トリエチレングリコールから選択される、上記［２２］に記載の方法。
［２４］ 前記キレート化部分が、式（ＩＩ）：

の構造又は式（ＩＩＩ）：

の構造を含む、上記［１６］に記載の方法。
［２５］ ２つの放射性金属イオンでポリペプチドを二重に標識する方法であって、
ａ．第１のクリック反応パートナー及び第２のクリック反応パートナーに共有結合した前記ポリペプチドを含む修飾ポリペプチドを提供することと
ｂ．キレート化部分に会合した前記第１の放射性金属イオンを含む第１の放射性錯体を提供することと、ここで、前記キレート化部分が、第３のクリック反応パートナーに共有結合したキレート剤を含み、
ｃ．キレート化部分に会合した前記第２の放射性金属イオンを含む第２の放射性錯体を提供することと、ここで前記キレート化部分が、第４のクリック反応パートナーに共有結合したキレート剤を含み、
ｄ．前記第１のクリック反応パートナーが前記第３のクリック反応パートナーと反応し、前記第２のクリック反応パートナーが前記第４のクリック反応パートナーと反応することによって、前記ポリペプチドを前記第１及び第２の放射性金属イオンで標識化することを可能にする条件下で、前記修飾ポリペプチドを前記放射性錯体と接触させることと、を含む、方法。
［２６］ 前記第１及び第２のクリック反応パートナーの一方がアルキン基を含み、前記第１及び第２のクリック反応パートナーの他方がアジドを含み、前記第３及び第４のクリック反応パートナーの一方がアルケン基を含み、前記第３及び第４のクリック反応パートナーの他方がジエンを含み、前記第１又は第２の放射性金属イオンが診断エミッタであり、前記他方が治療エミッタである、又は前記第１及び第２の放射性金属イオンの両方が治療エミッタである、上記［２５］に記載の方法。
［２７］ 上記［１］又は［１６］に記載の方法によって調製された放射性標識ポリペプチドと、薬学的に許容可能な担体とを含む、医薬組成物。
［２８］ 腫瘍性の疾患又は障害の治療を必要とする対象における、腫瘍性の疾患又は障害の治療方法であって、前記対象に、上記［２７］に記載の医薬組成物を投与することを含む、方法。
［２９］ 上記［１］又は［１６］に記載の方法によって調製された放射性標識抗体と、薬学的に許容可能な担体と、を含み、前記放射性標識抗体の免疫学的特性が保存されている、セラノスティック剤。
［３０］ 上記［１］に記載の方法によって調製されるセラノスティック剤であって、
式（ＶＩＩＩ）：

又は式（ＩＸ）：

の構造を有する、セラノスティック剤。
［３１］ 前記放射性金属イオンが、 ^３２ Ｐ、 ^４７ Ｓｃ、 ^６７ Ｃｕ、 ^７７ Ａｓ、 ^８９ Ｓｒ、 ^９０ Ｙ、 ^９９ Ｔｃ、 ^１０５ Ｒｈ、 ^１０９ Ｐｄ、 ^１１１ Ａｇ、 ^１３１ Ｉ、 ^１５３ Ｓｍ、 ^１５９ Ｇｄ、 ^１６５ Ｄｙ、 ^１６６ Ｈｏ、 ^１６９ Ｅｒ、 ^１７７ Ｌｕ、 ^１８６ Ｒｅ、 ^１８８ Ｒｅ、 ^１９４ Ｉｒ、 ^１９８ Ａｕ、 ^１９９ Ａｕ、 ^２１１ Ａｔ、 ^２１２ Ｐｂ、 ^２１２ Ｂｉ、 ^２１３ Ｂｉ、 ^２２３ Ｒａ、 ^２２５ Ａｃ、 ^２５５ Ｆｍ、 ^２２７ Ｔｈ、 ^６２ Ｃｕ、 ^６４ Ｃｕ、 ^６７ Ｇａ、 ^６８ Ｇａ、 ^８６ Ｙ、 ^８９ Ｚｒ、又は ^１１１ Ｉｎから選択される、上記［２９］に記載のセラノスティック剤。
［３２］ 組み合わせであって、
ａ．第１のクリック反応パートナーに共有結合したポリペプチドを含む修飾ポリペプチドと、
ｂ．キレート化部分に会合した放射性金属イオンを含む放射性錯体であって、前記キレート化部分が、第２のクリック反応パートナーに共有結合したキレート剤を含む、放射性錯体と、を含み、
前記組み合わせが、前記ポリペプチドを前記放射性金属イオンで標識するために使用される、組み合わせ。 All references (including patent applications, patents or publications) cited in this specification are by reference in their entirety to indicate that each individual publication or patent or patent application is incorporated in its entirety for all purposes. incorporated herein for all purposes to the same extent as if specifically and individually indicated to be incorporated by reference.

The following aspects may be included .
[1] A method for labeling a polypeptide with a radioactive metal ion, comprising:
a. providing a modified polypeptide comprising said polypeptide covalently attached to a first click reaction partner;
b. providing a radioactive complex comprising said radiometal ion associated with a chelating moiety, wherein said chelating moiety comprises a chelating agent covalently bound to a second click reaction partner;
c. complexing the modified polypeptide with the radioactive metal ion under conditions that allow the first click reaction partner to react with the second click reaction partner to label the polypeptide with the radiometal ion; and contacting with.
[2] one of the first and second click reaction partners comprises an alkyne group and the other of the click reaction partners comprises an azide, or one of the first and second click reaction partners comprises an alkene group; , The method according to [1] above, wherein the other of the click reaction partners comprises a diene.
[3] The method of [1] or [2] above, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.
[4] The method of [3] above, wherein the antibody is an anti-PSMA monoclonal antibody.
[5] The method according to any one of [1] to [4] above, wherein the radioactive metal ion is ²²⁵ Ac, ¹¹¹ In, or ⁸⁹ Zr.
[6] Any of the above [1] to [4], further comprising reacting an electrophile on the side chain with a sulfhydryl group covalently bonded to the first click reaction partner to obtain the modified polypeptide. or the method described in paragraph 1.
[7] The method according to any one of [1] to [5] above, wherein the modified polypeptide is a modified antibody or an antigen-binding fragment thereof obtained by site-specific incorporation of the first click reaction partner. .
[8] the modified antibody or antigen-binding fragment thereof trims the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for β-1,4 linkages between the core GlcNac residues of the Fc-glycosylation site of the antibody; to obtain a trimmed antibody or antigen-binding fragment thereof, and reacting the trimmed antibody or antigen-binding fragment thereof with an azide-labeled sugar in the presence of a glycosyltransferase such as GalT galactosyltransferase or GalNac transferase thereby obtaining the modified antibody or antigen-binding fragment thereof.
[9] The method according to [8] above, wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido-6-deoxyGalNAc.
[10] The method of [8] above, wherein the glycosyltransferase is GalT galactosyltransferase or GalNAc transferase.
[11] said modified antibody or antigen-binding fragment thereof deglycosylate the antibody or antigen-binding fragment thereof with an amidase to obtain a deglycosylated antibody or antigen-binding fragment thereof; reacting the fragment with azidoamine in the presence of microbial transglutaminase to obtain said modified polypeptide.
[12] The azidoamine is 3-azidopropylamine, 6-azidohexylamine, O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O The method according to [11] above, which is selected from '-(2-azidoethyl)pentaethylene glycol and O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol.
[13] The method of [1] above, wherein the modified polypeptide comprises the polypeptide covalently bound to an azide, tetrazine, or tetrazole group, either directly or via a linker.
[14] The chelating agent has the formula (I):

(wherein R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X,
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl,
Z is (CH2) _n Y;
n is 1 to 10,
Y is an electrophilic or nucleophilic moiety covalently attached to said second click reaction partner;
Alternatively, Z is hydrogen; and
R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X;
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl, or an electrophilic or nucleophilic moiety covalently bound to said second click reaction partner;
Alternatively, the method according to [1] above, wherein the chelating agent contains an open-chain ligand.
[15] The chelating moiety has the formula (II):

or formula (III):

The method according to [1] above, comprising the structure of
[16] A method of labeling an antibody or antigen-binding fragment thereof with ²²⁵ AC , ¹¹¹ In, or ⁸⁹ Zr _, comprising:
a. providing a modified antibody or antigen-binding fragment thereof comprising an antibody or antigen-binding fragment thereof covalently attached to an azide, tetrazine, or tetrazole group;
b. providing a radioactive complex comprising 225 AC, 111 In or 89 Zr associated with a chelating moiety, wherein said chelating moiety comprises a chelating agent covalently bound to ^an alkyne or ^alkene group ^; _and
c. under conditions that allow the azide, tetrazine, or tetrazole group to react with the alkyne or alkene group to label the antibody or antigen-binding fragment thereof with 225 AC , ₁₁₁ ^In , or ⁸⁹ Zr ^. contacting the modified antibody or antigen-binding fragment thereof with a radioactive complex;
The chelating agent has the formula (I):

(wherein R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X,
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl,
Z is (CH2) _n Y;
n is 1 to 10,
Y is an electrophilic or nucleophilic moiety covalently attached to the alkyne group;
Alternatively, Z is hydrogen; and
R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X;
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl, or an electrophilic or nucleophilic moiety covalently bonded to said alkyne group;
Alternatively, the method, wherein the chelating agent comprises an open-chain ligand.
[17] The above-described [16], further comprising reacting an electrophile on a side chain with a sulfhydryl group covalently bonded to the azide, tetrazine or tetrazole group to obtain the modified antibody or antigen-binding fragment thereof. the method of.
[18] The method of [16] above, wherein the modified antibody or antigen-binding fragment thereof is obtained by site-specific incorporation of the first click reaction partner.
[19] wherein the modified antibody or antigen-binding fragment thereof trims the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for β-1,4 linkages between core GlcNac residues in the Fc-glycosylation site of the antibody; to obtain a trimmed antibody or antigen-binding fragment thereof, and reacting the trimmed antibody or antigen-binding fragment thereof with an azide-labeled sugar in the presence of a glycosyltransferase to produce a modified antibody or antigen thereof obtaining a binding fragment. The method according to [18] above.
[20] The method of [19] above, wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido-6-deoxyGalNAc.
[21] The method of [19] above, wherein the glycosyltransferase is selected from GalT galactosyltransferase or GalNAc transferase.
[22] said modified antibody or antigen-binding fragment thereof deglycosylate the antibody or antigen-binding fragment thereof with an amidase to obtain a deglycosylated antibody or antigen-binding fragment thereof, and said deglycosylated antibody or antigen-binding fragment thereof; reacting the fragment with azidoamine in the presence of microbial transglutaminase to obtain said modified polypeptide.
[23] The azidoamine is 3-azidopropylamine, 6-azidohexylamine, O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O The method according to [22] above, which is selected from '-(2-azidoethyl)pentaethylene glycol and O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol.
[24] the chelating moiety is of formula (II):

The structure of or formula (III):

The method according to [16] above, comprising the structure of
[25] A method of dual labeling a polypeptide with two radioactive metal ions, comprising:
a. providing a modified polypeptide comprising said polypeptide covalently attached to a first click reaction partner and a second click reaction partner;
b. providing a first radioactive complex comprising said first radiometal ion associated with a chelating moiety, wherein said chelating moiety comprises a chelating agent covalently bound to a third click reaction partner;
c. providing a second radioactive complex comprising said second radioactive metal ion associated with a chelating moiety, wherein said chelating moiety comprises a chelating agent covalently bound to a fourth click reaction partner;
d. The first click reaction partner reacts with the third click reaction partner and the second click reaction partner reacts with the fourth click reaction partner, thereby combining the polypeptide with the first and second click reaction partners. contacting the modified polypeptide with the radioactive complex under conditions that allow labeling with the radioactive metal ion of
[26] one of the first and second click reaction partners comprises an alkyne group, the other of the first and second click reaction partners comprises an azide, and one of the third and fourth click reaction partners comprises an alkene group, the other of said third and fourth click reaction partners comprises a diene, said first or second radiometal ion is a diagnostic emitter and said other is a therapeutic emitter, or said The method of [25] above, wherein both the first and second radioactive metal ions are therapeutic emitters.
[27] A pharmaceutical composition comprising a radiolabeled polypeptide prepared by the method of [1] or [16] above and a pharmaceutically acceptable carrier.
[28] A method for treating a neoplastic disease or disorder in a subject in need thereof, comprising administering the pharmaceutical composition of [27] above to the subject. including, method.
[29] comprising a radiolabeled antibody prepared by the method of [1] or [16] above and a pharmaceutically acceptable carrier, wherein the immunological properties of the radiolabeled antibody are preserved; , theranostic agents.
[30] A theranostic agent prepared by the method described in [1] above,
Formula (VIII):

or formula (IX):

A theranostic agent having a structure of
[31] The radioactive metal ion is ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁷⁷ As , 89 Sr , ⁹⁰ Y , ⁹⁹ Tc, ¹⁰⁵ Rh, 109 ^Pd , ¹¹¹ Ag, ¹³¹ I , ¹⁵³ Sm, ^{159 Gd} , ¹⁶⁵ Dy , ¹⁶⁶ Ho, ^{169 Er} , ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁴ Ir, 198 Au , ¹⁹⁹ Au, ²¹¹ At , ²¹² Pb, ²¹² Bi , ²¹³ Bi, ²²³ Ra, ²²⁵ Ac, ²⁵⁵ Fm, ²²⁷ Th, ⁶² The theranostic agent according to [29] above, which is selected from Cu, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, or ¹¹¹ In.
[32] A combination comprising:
a. a modified polypeptide comprising a polypeptide covalently attached to a first click reaction partner;
b. a radioactive complex comprising a radioactive metal ion associated with a chelating moiety, said chelating moiety comprising a chelating agent covalently bound to a second click reaction partner;
A combination, wherein said combination is used to label said polypeptide with said radioactive metal ion.

Claims (32)

A method of labeling a polypeptide with a radioactive metal ion, comprising:
a. providing a modified polypeptide comprising said polypeptide covalently attached to a first click reaction partner;
b. providing a radioactive complex comprising said radiometal ion associated with a chelating moiety, wherein said chelating moiety comprises a chelating agent covalently bound to a second click reaction partner;
c. complexing the modified polypeptide with the radioactive metal ion under conditions that allow the first click reaction partner to react with the second click reaction partner to label the polypeptide with the radiometal ion; and contacting with. one of said first and second click reaction partners comprises an alkyne group and the other of said click reaction partners comprises an azide, or one of said first and second click reaction partners comprises an alkene group and said click 2. The method of claim 1, wherein the other of the reaction partners comprises a diene. 3. The method of claim 1 or 2, wherein said polypeptide is an antibody or antigen-binding fragment thereof. 4. The method of claim 3, wherein said antibody is an anti-PSMA monoclonal antibody. Claims 1-1, wherein the radioactive metal ion is ²²⁵ Ac, ¹¹¹ In, or ⁸⁹ Zr
5. The method of any one of 4. 5. The method according to any one of claims 1 to 4, further comprising reacting an electrophile on a side chain with a sulfhydryl group covalently attached to said first click reaction partner to obtain said modified polypeptide. Method. A method according to any one of claims 1 to 5, wherein said modified polypeptide is a modified antibody or antigen-binding fragment thereof obtained by site-specific incorporation of said first click reaction partner. said modified antibody or antigen-binding fragment thereof trimming the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for β-1,4 linkages between core GlcNac residues of the Fc-glycosylation site of said antibody; Obtaining a trimmed antibody or antigen-binding fragment thereof and GalT galactosyltransferase or GalNa
reacting the trimmed antibody or antigen-binding fragment thereof with an azide-labeled sugar in the presence of a glycosyltransferase such as c-transferase to obtain the modified antibody or antigen-binding fragment thereof. 8. The method of claim 7. The azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalN
az) or UDP-6-azido-6-deoxyGalNAc. 9. The method of claim 8, wherein said glycosyltransferase is GalT galactosyltransferase or GalNAc transferase. said modified antibody or antigen-binding fragment thereof deglycosylating the antibody or antigen-binding fragment thereof with an amidase to obtain a deglycosylated antibody or antigen-binding fragment thereof; 8. A method according to claim 7, obtained by a method comprising obtaining said modified polypeptide by reacting with an azidoamine in the presence of a microbial transglutaminase. The azidoamines include 3-azidopropylamine, 6-azidohexylamine, O-(
2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2
-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, and O-(
12. The method of claim 11, selected from 2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol. 2. The method of claim 1, wherein said modified polypeptide comprises said polypeptide covalently attached, either directly or through a linker, to an azide, tetrazine, or tetrazole group. The chelating agent has the formula (I):

(wherein R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X,
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl,
Z is (CH2) _n Y;
n is 1 to 10,
Y is an electrophilic or nucleophilic moiety covalently attached to said second click reaction partner;
Alternatively, Z is hydrogen; and R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X,
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl, or an electrophilic or nucleophilic moiety covalently attached to said second click reaction partner;
Alternatively, the chelating agent comprises an open chain ligand)
2. The method of claim 1, comprising a macrocycle having the structure of The chelating moiety has the formula (II):

or formula (III):

2. The method of claim 1, comprising the structure of A method of labeling an antibody or antigen-binding fragment thereof with ²²⁵ _AC , ¹¹¹ In, or ⁸⁹ Zr, comprising:
a. providing a modified antibody or antigen-binding fragment thereof comprising an antibody or antigen-binding fragment thereof covalently attached to an azide, tetrazine, or tetrazole group;
b. providing a radioactive complex comprising ²²⁵ _AC , ¹¹¹ In or ⁸⁹ Zr associated with a chelating moiety, wherein said chelating moiety comprises a chelating agent covalently bound to an alkyne or alkene group; and c. The azide, tetrazine, or tetrazole group reacts with the alkyne or alkene group to render the antibody or antigen-binding fragment thereof ²²⁵ _AC , ¹¹¹ In
or, contacting the modified antibody or antigen-binding fragment thereof with a radioactive complex under conditions that allow labeling with ^89Zr ;
The chelating agent has the formula (I):

(wherein R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X,
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl,
Z is (CH2) _n Y;
n is 1 to 10,
Y is an electrophilic or nucleophilic moiety covalently attached to the alkyne group;
Alternatively, Z is hydrogen; and R ₁ , R ₂ , R ₃ and R ₄ are each independently CHQCO ₂ X,
Q is independently hydrogen, C ₁ -C ₄ alkyl or (C ₁ -C ₂ alkyl)phenyl;
X is independently hydrogen, benzyl, C ₁ -C ₄ alkyl, or an electrophilic or nucleophilic moiety covalently bonded to said alkyne group;
Alternatively, the chelating agent comprises an open chain ligand)
A method, including the structure of 17. The method of claim 16, further comprising reacting an electrophile on a side chain with a sulfhydryl group covalently attached to said azide, tetrazine or tetrazole group to obtain said modified antibody or antigen-binding fragment thereof. 17. The method of claim 16, wherein said modified antibody or antigen-binding fragment thereof is obtained by site-specific incorporation of said first click reaction partner. said modified antibody or antigen-binding fragment thereof trimming the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for β-1,4 linkages between core GlcNac residues of the Fc-glycosylation site of said antibody; obtaining a trimmed antibody or antigen-binding fragment thereof, and reacting the trimmed antibody or antigen-binding fragment thereof with an azide-labeled sugar in the presence of a glycosyltransferase to produce a modified antibody or antigen-binding fragment thereof; 19. The method of claim 18, obtained by a method comprising obtaining. The azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalN
az) or UDP-6-azido-6-deoxyGalNAc. 20. The method of claim 19, wherein said glycosyltransferase is selected from GalT galactosyltransferase or GalNAc transferase. said modified antibody or antigen-binding fragment thereof deglycosylating the antibody or antigen-binding fragment thereof with an amidase to obtain a deglycosylated antibody or antigen-binding fragment thereof; 17. A method according to claim 16, obtained by a method comprising obtaining said modified polypeptide by reacting with an azidoamine in the presence of a microbial transglutaminase. The azidoamines include 3-azidopropylamine, 6-azidohexylamine, O-(
2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2
-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, and O-(
23. The method of claim 22, selected from 2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol. The chelating moiety has the formula (II):

The structure of or formula (III):

17. The method of claim 16, comprising the structure of A method of dual labeling a polypeptide with two radioactive metal ions, comprising:
a. providing a modified polypeptide comprising said polypeptide covalently attached to a first click reaction partner and a second click reaction partner; b. providing a first radioactive complex comprising said first radiometal ion associated with a chelating moiety, wherein said chelating moiety comprises a chelating agent covalently bound to a third click reaction partner;
c. providing a second radioactive complex comprising said second radioactive metal ion associated with a chelating moiety, wherein said chelating moiety comprises a chelating agent covalently bound to a fourth click reaction partner;
d. The first click reaction partner reacts with the third click reaction partner and the second click reaction partner reacts with the fourth click reaction partner, thereby combining the polypeptide with the first and second click reaction partners. contacting the modified polypeptide with the radioactive complex under conditions that allow labeling with the radioactive metal ion of one of the first and second click reaction partners comprises an alkyne group, the other of the first and second click reaction partners comprises an azide, and one of the third and fourth click reaction partners comprises an alkene group. wherein the other of said third and fourth click reaction partners comprises a diene, said first or second radioactive metal ion is a diagnostic emitter and said other is a therapeutic emitter, or said first and fourth 26. The method of claim 25, wherein both of the two radioactive metal ions are therapeutic emitters. 17. A pharmaceutical composition comprising a radiolabeled polypeptide prepared by the method of claim 1 or 16 and a pharmaceutically acceptable carrier. 28. A method of treating a neoplastic disease or disorder in a subject in need thereof, comprising administering to said subject the pharmaceutical composition of claim 27. 17. A theranostic agent comprising a radiolabeled antibody prepared by the method of claim 1 or 16 and a pharmaceutically acceptable carrier, wherein the immunological properties of said radiolabeled antibody are preserved. A theranostic agent prepared by the method of claim 1,
Formula (VIII):

or formula (IX):

A theranostic agent having a structure of The radioactive metal ion is ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁷⁷ As, ⁸⁹ Sr, ⁹⁰ Y
, ⁹⁹ Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹³¹ I, ¹⁵³ Sm, ¹⁵⁹ Gd
, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁴
Ir, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹² Pb, ²¹² Bi, ²¹³ Bi, ^2
23 Ra, ²²⁵ Ac, ²⁵⁵ Fm, ²²⁷ Th, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸
30. The theranostic agent of claim 29, selected from Ga, ^<86> Y, ^<89> Zr, or ^<111> In. is a combination of
a. a modified polypeptide comprising a polypeptide covalently attached to a first click reaction partner;
b. a radioactive complex comprising a radioactive metal ion associated with a chelating moiety, said chelating moiety comprising a chelating agent covalently bound to a second click reaction partner;
A combination, wherein said combination is used to label said polypeptide with said radioactive metal ion.

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